Highly active transposase proteins of transposon systems and uses thereof

Abstract

The present invention relates to highly active transposase proteins of a transposon system and uses thereof. According to the present invention, genes can be effectively transferred by improving transposase activity, and thus the present invention can be effectively used for developing genome-modified cell lines that express various genes. According to the present invention, when a T cell is converted using a highly active transposase, the proportion of cytotoxic T (CD8 + T) cells having anti-cancer activity is increased, and the proportion of memory-type T cells of TCM (central memory T cells) and TSCM (stem cell-like memory T cells) having good durability in vivo is increased, and the T cell conversion efficiency is improved. Therefore, it is expected that TCR-T cells and CAR-T cells having excellent persistence in vivo can be produced using the transposon system of the present invention.

Description

Technical Field

[0001] This invention relates to highly active transposase proteins of transposon systems and their uses.

[0002] This application claims priority and benefit to Korean Patent Application No. 10-2023-0014296, filed on February 2, 2023, the disclosure of which is incorporated herein by reference in its entirety. Background Technology

[0003] Chimeric antigen receptor (CAR)-T cells are a type of cell therapy that inserts an antibody sequence that binds to a tumor antigen (such as CD19) into T cells, along with domains required for T cell signaling (such as CD3 / 4-1BB / CD28). There are many methods for inserting these CAR genes into T cells, but most use lentiviral delivery systems. Lentivirals are characterized by their ability to continuously express genes because they integrate into the cell's chromosome. The production cost of these lentiviruses is high, a major factor contributing to the rising price of therapeutics, but their advantage is that once produced, they can be used for multiple patients.

[0004] On the other hand, personalized T-cell receptor-modified T-cell therapy (TCR-T) is generated by identifying TCR sequences that react with neoantigens present in each patient and delivering these sequences into T cells via a gene delivery system. However, because it is personalized, the TCR sequences applied to each patient are different, making it nearly impossible to apply to lentiviruses. Therefore, it is necessary to develop TCR-T cells using transposons, which are non-viral vectors that are easier to produce than lentiviruses, have lower production costs, and can be integrated into chromosomes for continuous gene expression.

[0005] Therefore, in order to meet the above requirements, the inventors have developed a transposon as a gene delivery vector that can insert exogenous genes into the chromosomes (genome) of target cells, especially immune cells (Korean Patent Publication No. 10-2602485).

[0006] Furthermore, as a result of extensive research on improving gene delivery efficiency through transposon systems, the inventors increased the activity of transposases in transposon systems and confirmed that they exhibited excellent gene transfer efficiency, thus completing this invention. Summary of the Invention

[0007] [Technical Issues]

[0008] The inventors completed this invention by confirming that when the activity of transposase is improved by optimizing the nucleic acid sequence of the transposase, the gene delivery efficiency through the transposon system is increased.

[0009] The purpose of this invention is to provide a transposase expression vector containing a nucleic acid sequence encoding a transposase represented by SEQ ID NO:2 or SEQ ID NO:3.

[0010] Another object of the present invention is to provide an mRNA encoding a transposase represented by SEQ ID NO:12.

[0011] Another object of the present invention is to provide a transposase expressed by the transposase expression vector of the present invention or by mRNA encoding the transposase of the present invention.

[0012] Another object of the present invention is to provide a transposon system for delivering target DNA, comprising: a) a transposon vector wherein the target DNA is inserted; and b) a transposase expression vector comprising a nucleic acid sequence encoding a transposase represented by SEQ ID NO:2 or SEQ ID NO:3, or mRNA encoding a transposase represented by SEQ ID NO:12, or the transposase of the present invention.

[0013] Another object of the present invention is to provide a transposon kit for delivering target DNA, comprising a transposon system for delivering target DNA according to the present invention and its instructions.

[0014] Another object of the present invention is to provide a cell in which: a) A transposon vector containing inserted target DNA; and b) A transposase expression vector containing a nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 or SEQ ID NO:3, or mRNA encoding the transposase represented by SEQ ID NO:12, or The transposase of the present invention.

[0015] Another object of the present invention is to provide a method for inserting a target DNA sequence into a cell genome, comprising the steps of introducing a) and b) into the cell: a) A transposon vector containing inserted target DNA; and b) A transposase expression vector containing a nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 or SEQ ID NO:3, or mRNA encoding the transposase represented by SEQ ID NO:12, or The transposase of the present invention.

[0016] However, the technical problems to be solved by the present invention are not limited to those described above, and those skilled in the art will fully understand other problems not described herein through the following description.

[0017] [Technical Solution]

[0018] To achieve the above objectives, the present invention provides a transposase expression vector comprising a nucleic acid sequence encoding a transposase represented by SEQ ID NO:2 or SEQ ID NO:3.

[0019] In one embodiment of the present invention, the nucleic acid sequence encoding the transposase may further include a nucleic acid sequence encoding a nuclear localization signal (NLS), but is not limited thereto.

[0020] In another embodiment of the invention, the nucleic acid sequence encoding the nuclear localization signal may be one or more of the nucleic acid sequences represented by SEQ ID NO:4 to 11, but is not limited thereto.

[0021] In another embodiment of the present invention, the nucleic acid sequence encoding the nuclear localization signal may be included at the 5' end or 3' end of the nucleic acid sequence encoding the transposase along the 5' to 3' direction, but is not limited thereto.

[0022] In addition, the present invention provides an mRNA encoding a transposase represented by SEQ ID NO:12.

[0023] In one embodiment of the present invention, mRNA can be generated by in vitro transcription using the transposase expression vector of the present invention, but is not limited thereto.

[0024] In another embodiment of the present invention, the transposase expression vector may be a transposase expression vector containing a nucleic acid sequence encoding the transposase represented by SEQ ID NO:3, but is not limited thereto.

[0025] Furthermore, the present invention provides a transposase expressed by the transposase expression vector of the present invention or by mRNA encoding the transposase of the present invention.

[0026] In one embodiment of the present invention, when the transposase expression vector is a transposase expression vector containing a nucleic acid sequence encoding the transposase represented by SEQ ID NO:3, the transposase can be expressed by mRNA prepared by in vitro transcription using the transposase expression vector, but is not limited thereto.

[0027] In another embodiment of the invention, the transposase may include, but is not limited to, the amino acid sequence represented by SEQ ID NO:13.

[0028] Furthermore, the present invention provides a transposon system for delivering target DNA, comprising: a) a transposon vector in which target DNA is inserted; and b) a transposase expression vector comprising a nucleic acid sequence encoding a transposase represented by SEQ ID NO:2 or SEQ ID NO:3, or mRNA encoding a transposase represented by SEQ ID NO:12, mRNA encoding the transposase of the present invention, or the transposase of the present invention.

[0029] In one embodiment of the invention, the transposon vector and the 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 are not limited thereto.

[0030] Furthermore, the present invention provides a transposon kit for delivering target DNA, comprising a transposon system for delivering target DNA according to the present invention and its instructions.

[0031] Furthermore, the present invention provides a cell in which:

[0032] a) A transposon vector containing inserted target DNA; and

[0033] b) A transposase expression vector containing a nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 or SEQ ID NO:3, or mRNA encoding the transposase represented by SEQ ID NO:12, or mRNA encoding the transposase of the present invention, or The transposase of the present invention.

[0034] In one embodiment of the present invention, the target DNA can be cleaved from the transposon vector by an intracellular transposase, and the cleaved target DNA can be inserted into the genome of the cell, but is not limited thereto.

[0035] In another embodiment of the invention, the cells may be selected from a group consisting of T cells, NK cells, B cells, dendritic cells, macrophages and mast cells, but are not limited thereto.

[0036] In another embodiment of the present invention, after the transposon vector is introduced, the cells can be co-cultured with feeder cells, but is not limited thereto.

[0037] In another embodiment of the invention, the feeder cells may be cells irradiated with radiation, but are not limited thereto.

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

[0039] Furthermore, the present invention provides a method for inserting a target DNA sequence into a cell genome, comprising the steps of introducing a) and b) into the cell: a) A transposon vector containing inserted target DNA; and b) A transposase expression vector containing a nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 or SEQ ID NO:3, or mRNA encoding the transposase represented by SEQ ID NO:12, or mRNA encoding the transposase of the present invention, or The transposase of the present invention.

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

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

[0042] In another embodiment of the invention, the co-culture step with feeder cells can be performed immediately after the introduction step, but is not limited thereto.

[0043] [Beneficial Effects]

[0044] This invention relates to highly active transposase proteins of transposon systems and their uses, and can be effectively applied to the development of genome-modified cell lines expressing various genes, as genes can be efficiently delivered by increasing the activity of transposases.

[0045] Furthermore, when T cells are transduced using the highly active transposase according to the present invention, cytotoxic T (CD8) cells with anticancer activity are produced. + The ratio of T cells increases, and memory T cells (such as T cells) with excellent in vivo persistence increase. CM (Central memory T cells) and T SCM The ratio of stem cell-like memory T cells is increased. Therefore, it is expected that the transposon system of the present invention can be used to produce TCR-T cells and CAR-T cells with excellent in vivo persistence. Attached Figure Description

[0046] Figures 1 to 3 The following groups were shown on day 7 after electroporation ( Figure 1 Day 10 Figure 2 ) and the 14th day ( Figure 3Expression of 1G4 TCR in T cells: including groups that underwent only electroporation (EP) without plasmids in PBMCs (EP only); groups that introduced pBat transposons containing the 1G4 TCR gene and existing transposases (existing transposases); and groups that introduced pBat transposons containing the 1G4 TCR gene and optimized transposases (optimized transposases).

[0047] Figure 4 The following groups were compared at days 7, 10, and 14 post-electroporation: the group that underwent electroporation (EP) without plasmids in PBMCs (EP only); the group that introduced a pBat transposon containing the 1G4 TCR gene and an existing transposase (existing transposase); and the group that introduced a pBat transposon containing the 1G4 TCR gene and an optimized transposase (optimized transposase).

[0048] Figure 5 The following groups showed CD4 expression in T cells of 1G4TCR on days 7, 10, and 14 after electroporation. + and CD8 + Cell populations: including groups that only underwent electroporation (EP) and had no plasmids in PBMCs (EP only); groups that introduced pBat transposons containing the 1G4TCR gene and existing transposases (existing transposases); and groups that introduced pBat transposons containing the 1G4TCR gene and optimized transposases (optimized transposases).

[0049] Figure 6 The following groups showed CD4 expression in T cells of 1G4TCR on days 7, 10, and 14 after electroporation. + and CD8 + Comparison of cell populations: the group that introduced the pBat transposon containing the 1G4 TCR gene and the existing transposase (existing transposase); and the group that introduced the pBat transposon containing the 1G4 TCR gene and the optimized transposase (optimized transposase).

[0050] Figure 7 The following groups were shown to identify memory T cells in 1G4 TCR-expressing T cells using CD45RA and CCR7 markers at days 7, 10, and 14 post-electroporation: the group that underwent electroporation (EP) alone without plasmids in PBMCs (EP only); the group that introduced a pBat transposon containing the 1G4TCR gene and an existing transposase (existing transposase); and the group that introduced a pBat transposon containing the 1G4 TCR gene and an optimized transposase (optimized transposase).

[0051] Figures 8a to 10bThe images show Jurkat cells on day 1 after electroporation using pBat transposons containing the GFP gene and existing transposases (P5081 + existing transposase) or pBat transposons containing the GFP gene and optimized transposases (P5081 + optimized transposase). Figure 8a and Figure 8b Day 7 Figure 9a and Figure 9b ) and the 14th day ( Figure 10a and Figure 10b FACS analysis results of GFP expression level.

[0052] Figure 11 The results of PCR amplification of transposase genes optimized for mRNA format are shown.

[0053] Figure 12 The enzyme map results used to confirm the structure are shown after infusion cloning between the mRNA template (vector) and transposase (insertion) to construct the mRNA template plasmid.

[0054] Figure 13 The total number of 1G4 TCR-T cells produced according to the transposase gene delivery pattern is shown.

[0055] Figure 14 The survival rate of 1G4 TCR-T cells generated according to the transposase gene delivery form is shown.

[0056] Figure 15 A FACS analysis method for comparing GFP gene expression based on the form of transposase gene delivery after electroporation is shown.

[0057] Figure 16 The results of FACS analysis based on GFP gene expression in transposase form after electroporation are shown.

[0058] Figure 17 A FACS analysis method is shown for comparing 1G4TCR gene expression based on the form of transposase gene delivery after electroporation.

[0059] Figure 18 The expression of the 1G4 TCR gene according to the form of transposase after electroporation is shown.

[0060] Figure 19 The classification of memory T cells based on CD45RO / CD62L marker expression is shown.

[0061] Figure 20 The proportion of memory T cells in T cells expressing 1G4 TCR based on transposase gene delivery form after electroporation is shown.

[0062] Figure 21The ratio of CD4 to CD8 in T cells expressing 1G4 TCR based on transposase gene delivery form after electroporation is shown.

[0063] Figure 22 and Figure 23 This demonstrates the use of a cloning template to add the NLS (nuclear localization sequence) gene to the 5' end of an optimized transposase. Figure 22 ) or 3' end ( Figure 23 The various parts of the synthetic sequence of ).

[0064] Figures 24a to 26 This shows day 1 after transfection (electroporation) with an optimized transposase containing the NLS (nuclear localization sequence) gene at its 5' or 3' end. Figure 24a and Figure 24b Day 7 Figure 25a and Figure 25b ) and the 14th day ( Figure 26 The results of evaluating GFP gene delivery efficiency.

[0065] Figure 27 The figure shows the FACS analysis results of GFP gene expression on day 1 and day 7 after transfection (electroporation) with an optimized transposase containing an NLS (nuclear localization sequence) gene at its 5' or 3' end.

[0066] Figure 28 The figure shows the FACS analysis results of high-intensity GFP gene expression on day 1 and day 7 after transfection (electroporation) with an optimized transposase containing an NLS (nuclear localization sequence) gene at its 5' or 3' end.

[0067] Figure 29a and Figure 29b The results show that on day 14 after transfection (electroporation) with an optimized transposase containing the NLS (nuclear localization sequence) gene at its 5' or 3' end, cells expressing GFP were sorted on day 15, and the sorted cells were cultured for an additional 9 days to evaluate the GFP gene delivery efficiency.

[0068] Figures 30a to 31d This shows the results on day 7 after transposase transfection (electroporation) with the NLS (nuclear localization sequence) gene added to its 5' or 3' end. Figures 30a to 30d ) and the 14th day ( Figures 31a to 31d The results of evaluating the efficiency of 1G4 TCR gene delivery and the CD4 / CD8 ratio in 1G4 TCR-expressing T cells were presented.

[0069] Figure 32 The structure of the CD19 CAR vector of transposons and lentiviruses is shown.

[0070] Figure 33 The co-culture conditions used for in vitro killing analysis are shown.

[0071] Figure 34 The results show the enzyme map that confirms the structure of the pBat CD19 CAR transposon plasmid vector.

[0072] Figure 35 and Figure 36 The figures show the total number of T cells cultured after gene delivery into LK032 PBMCs and LK053 PBMCs via transduction and electroporation using lentivirus and transposon, respectively, to generate CD19 CAR-T cells, and after culturing the cells for 7 and 14 days. Figure 35 ) and cell viability ( Figure 36 ).

[0073] Figure 37 This demonstrates a FACS analysis method for comparing CD19CAR protein expression based on gene delivery vectors (lentivirus or transposon).

[0074] Figure 38 The results show the assessment of CD19 CAR expression in LK032PBMCs on days 7 and 14, based on the gene delivery vector (lentivirus or transposon).

[0075] Figure 39 The results show the assessment of CD19 CAR expression in LK053PBMCs on days 7 and 14, based on the gene delivery vector (lentivirus or transposon).

[0076] Figure 40 The classification of memory T cells based on CD45RA / CD62L marker expression is shown.

[0077] Figure 41 The results show the proportion of memory T cells in T cells from LK032 PBMCs, based on the gene delivery vector (lentivirus or transposon).

[0078] Figure 42 The results show the proportion of memory T cells in CD19CAR-expressing T cells from LK053 PBMCs, based on the gene delivery vector (lentivirus or transposon).

[0079] Figure 43 The results show the proportion of memory T cells in CD19 CAR-expressing T cells from LK032 PBMC and LK053 PBMC, based on the gene delivery vector (lentivirus or transposon).

[0080] Figure 44 and Figure 45The study showed the evaluation of CD19 CAR-expressing T cell intracellular CD4 based on gene delivery vector (lentivirus or transposon). + -Expressing T cells ( Figure 44 ) and CD8 + -Expressing T cells ( Figure 45 The result of the proportion.

[0081] Figure 46 and Figure 47 It shows the use of lentivirus ( Figure 46 ) or transposable Figure 47 The average killing percentage of FLAG (CD19-CAR)-expressing T cells transduced as gene delivery vectors. Detailed Implementation

[0082] The inventors completed this invention by confirming that when the activity of transposase is enhanced by optimizing the nucleic acid sequence of the transposase, the gene delivery efficiency through the transposon system is improved.

[0083] The present invention will be described in detail below.

[0084] The present invention provides a transposase expression vector comprising a nucleic acid sequence encoding a transposase represented by SEQ ID NO:2 or SEQ ID NO:3.

[0085] In this invention, the term "transposase" refers to an enzyme that recognizes and binds to both ends of a transposon (particularly inverted repeat sequences), cuts the region, and then moves the gene fragment between the two ends (i.e., the region containing the target DNA) to another location in the chromosome.

[0086] Transposases can be introduced into cells either as the protein itself, or as a vector containing a sequence encoding the transposase protein (“transposase vector”, “transposase plasmid”, “transposon expression vector”, or “transposase expression plasmid”) or as a nucleic acid molecule (DNA or RNA molecule) containing a sequence encoding the transposase protein, and can be expressed in the cells after introduction.

[0087] In this invention, the term "vector" refers to a nucleic acid molecule capable of delivering another linked nucleic acid molecule. Specifically, a vector is any carrier used to introduce and / or transfer nucleotides into and / or into host cells in vitro, ex vivo, or in vivo, and can be a replicon that, when bound to another DNA fragment, causes the replication of the linked fragment. The term "replicon" refers to any genetic unit that functions autonomously as a unit of DNA replication in vivo, i.e., a genetic unit capable of replicating under its own control, such as plasmids, bacteriophages, granules, chromosomes, or viruses. Vectors can include, but are not limited to, bacteria, plasmids, bacteriophages, granules, free organisms, viruses, or insertable DNA fragments, i.e., fragments capable of inserting into the host cell genome through homologous recombination.

[0088] In this invention, the term "transposase expression vector" refers to a nucleic acid molecule capable of delivering a nucleic acid sequence encoding a transposase or a template nucleic acid sequence for generating mRNA encoding a transposase, and may be called a "transposase vector," "transposase plasmid," or "transposase expression plasmid."

[0089] According to one example of the present invention, a transposase expression vector containing a nucleic acid sequence encoding a transposase represented by SEQ ID NO:2 may contain a nucleic acid sequence of SEQ ID NO:14, and a nucleic acid sequence containing a transposase represented by SEQ ID NO:3 may contain a nucleic acid sequence of SEQ ID NO:15, but is not limited thereto.

[0090] The vector according to the invention can be double-stranded DNA in the form of plasmid DNA, linear DNA, hairpin DNA, or microcircular DNA, or it can be a recombinant viral vector, but is not limited thereto. It can be used without limitation as long as the vector contains a nucleic acid sequence encoding a transposase or a template nucleic acid sequence for generating mRNA encoding a transposase, and is capable of delivering that sequence to target cells, and those skilled in the art can choose from a variety of vectors known in the art.

[0091] The vector of the present invention may preferably include: a promoter as a transcription initiation factor for RNA polymerase binding, an optional operator sequence for regulating transcription, an enhancer sequence, a sequence encoding a suitable mRNA ribosome binding site, and sequences for regulating transcription and translation termination, such as terminators. 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, and a cloning site, and may also include additional genes, such as tag genes and selection marker genes, for example, genes for selecting antibiotic resistance in transformants. In the vector, the sequences of the various genes are operatively linked to the promoter. As used herein, the term "operatively linked" refers to a functional link between a nucleotide expression regulatory sequence (such as a promoter sequence) and another nucleotide sequence, whereby the regulatory sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0092] The vectors of this invention can be constructed using prokaryotic or eukaryotic cells as hosts. For example, when the vector of this invention is an expression vector and uses a prokaryotic cell as a host, it typically contains a strong promoter capable of initiating transcription (e.g., pLλ promoter, trp promoter, lac promoter, tac promoter, and T7 promoter), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. When a eukaryotic cell is used as a host, the vector may include, but is not limited to, eukaryotic origins of replication such as f1 origin, SV40 origin, pMB1 origin, adenovirus origin, AAV origin, or BBV origin. Furthermore, promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, or HSV-tk promoter) may be used, and the vector typically contains a polyadenylated sequence as a transcription termination sequence. The vector may also include signaling sequences such as poly A signals, but is not limited to these.

[0093] Examples of tag genes include, but are not limited to, Avi tags, calmodulin tags, polyglutamic acid tags, E tags, FLAG tags, HA tags, His tags (polyhistidine tags), myc tags, S tags, SBP tags, IgG-Fc tags, CTB tags, Softag 1 tags, Softag 3 tags, Strep tags, TC tags, V5 tags, VSV tags, and Xpress tags. Preferably, the vector according to the invention may contain a myc tag.

[0094] In this invention, the vector can be delivered to prokaryotic or eukaryotic host cells using various techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into cells. For example, the vector of this invention can be introduced into cells via: calcium phosphate coprecipitation; electroporation; microfluidic gene editing; nuclear infection; cell extrusion; acoustic perforation; optical transfection; puncture infection; gene gun; magnetic infection; viral transduction; DEAE-glucan transfection; lipid infection; or transfection using dendritic macromolecules, liposomes, or cationic polymers, but is not limited thereto.

[0095] According to one example of the invention, the nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 is a codon-optimized sequence, wherein the DNA sequence of an existing transposase (pBat transposase, SEQ ID NO:1) is optimized to enhance transposase expression, particularly in cells such as T cells, characterized in that it provides the transposase in the form of (plasmid) DNA, i.e., a vector containing a sequence encoding the transposase protein.

[0096] In this invention, the nucleic acid sequence encoding the transposase represented by SEQ ID NO:3 is an optimized sequence, wherein the DNA sequence of an existing transposase (pBat transposase, SEQ ID NO:1) is suitable for mRNA format, and according to one example of the invention, the nucleic acid sequence encoding the transposase represented by SEQ ID NO:3 is used to generate mRNA by in vitro transcription using a transposase expression vector containing this sequence, thereby providing the transposase 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 generating mRNA encoding the transposase.

[0097] In this invention, the nucleic acid sequence represented by SEQ ID NO:2 or SEQ ID NO:3 is optimized at the nucleic acid sequence level, and the transposase protein expressed therefrom is identical to existing transposase proteins.

[0098] In this invention, the term "nucleic acid" or "nucleic acid molecule" broadly includes DNA (gDNA and cDNA) and RNA molecules. Nucleotides, as the basic structural units of nucleic acids, include not only natural nucleotides but also analogs with modified sugar or base portions. The nucleotide sequence of the nucleic acid according to the invention can be modified, and such modifications include the addition, deletion, non-conservative substitution, or conserved substitution of nucleotides. The nucleic acid of the invention also includes a nucleotide sequence that is substantially identical to the nucleotide sequence described above. Substantial identity means that when compared with the nucleotide sequence of the invention to maximize correspondence and analyzed using algorithms commonly used in the art, the nucleotide sequence exhibits at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology.

[0099] In other words, in this invention, the polynucleotide composed of a nucleotide sequence represented by a specific sequence number is not limited to that sequence alone; variants of that sequence are also included within the scope of this invention. The nucleic acid molecules composed of nucleotide sequences represented by specific sequence numbers in this invention include functional equivalents of nucleic acid molecules, such as variants in which a portion of the nucleotide sequence of a nucleic acid molecule is modified by deletion, substitution, or insertion, but which are capable of performing the same function as the nucleic acid molecule. Specifically, the polynucleotides disclosed in this invention may include nucleotide sequences having at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% sequence identity with the nucleotide sequence represented by the specific sequence number. For example, polynucleotides include those with 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. The “sequence identity %” of a polynucleotide is determined by comparing a comparison region between two optimally aligned sequences. This comparison region may include portions of the polynucleotide sequence with insertions or deletions (i.e., gaps) relative to a reference sequence to achieve optimal alignment of the two sequences (excluding insertions or deletions).

[0100] According to one example of the invention, the nucleic acid sequence encoding the transposase may further include a nucleic acid sequence encoding the nuclear localization signal (NLS).

[0101] In this invention, "nuclear localization signal (NLS)" refers to a peptide chain that promotes the transport of various nuclear proteins from the cytoplasm to the nucleus. NLS is well known in the art and can be any known or functional NLS widely described in the literature. According to one example of the invention, the nuclear localization signal may be SV40 or C-myc.

[0102] In this invention, the nucleic acid sequence encoding the nuclear localization signal can be one or more of the nucleic acid sequences represented by SEQ ID NO:4 to 11. Specifically, SEQ ID NO:4 to 9 are sequences encoding SV40, and SEQ ID NO:10 to 11 are sequences encoding C-myc. The nuclear localization signal can be included in one or more copies, and can include a combination of two or more types of nuclear localization signals.

[0103] According to one example of the present invention, the nucleic acid sequence encoding the nuclear localization signal is also optimized. The optimized nucleic acid sequence of SV40 may contain the nucleotide sequence of SEQ ID NO:9, and the optimized nucleic acid sequence of C-myc may contain the nucleotide sequence of SEQ ID NO:11.

[0104] Since the optimization was performed at the nucleic acid sequence level, the SV40 protein expressed by SEQ ID NO:4 to 9 and the C-myc protein expressed by SEQ ID NO:10 to 11 are identical.

[0105] According to one example of the invention, the nucleic acid sequence encoding the nuclear localization signal may be included at the 5' or 3' end of the nucleic acid sequence encoding the transposase, in the 5' to 3' direction. Specifically, to enhance the activity of the transposase, when the SV40 or C-myc nucleic acid sequence is added to the 5' end of the transposase nucleic acid sequence, a Kozak sequence (5'-GCCACC-3') may be additionally included upstream of the start codon of the transposase, but is not limited thereto (see [reference]). Figure 22 ).

[0106] In this invention, the nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 (which has an optimized SV40 nucleic acid sequence (SEQ ID NO:9) added to its 5' end) may comprise or consist of the nucleic acid sequence of SEQ ID NO:16.

[0107] In this invention, the nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 (which has an optimized C-myc nucleic acid sequence (SEQ ID NO:11) added to its 5' end) may comprise or consist of the nucleic acid sequence of SEQ ID NO:18.

[0108] In this invention, the nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 (which has an optimized SV40 nucleic acid sequence (SEQ ID NO:9) added to its 3' end) may comprise or consist of the nucleic acid sequence of SEQ ID NO:20.

[0109] In this invention, the nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 (which has an optimized C-myc nucleic acid sequence (SEQ ID NO:11) added to its 3' end) may include or consist of the nucleic acid sequence of SEQ ID NO:22.

[0110] In this invention, the transposase expression vector containing the nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 may contain or consist of the nucleic acid sequence of SEQ ID NO:17, wherein an optimized SV40 nucleic acid sequence (SEQ ID NO:9) is added to the 5' end, and a Kozak sequence (5′-GCCACC-3′) is included upstream of the start codon of the transposase (see...). Figure 22 ).

[0111] In this invention, the transposase expression vector containing the nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 may contain or consist of the nucleic acid sequence of SEQ ID NO:19, wherein an optimized C-myc nucleic acid sequence (SEQ ID NO:11) is added to the 5' end, and a Kozak sequence (5′-GCCACC-3′) is included upstream of the start codon of the transposase (see...). Figure 22 ).

[0112] In this invention, the transposase expression vector comprising the nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 (which has an optimized SV40 nucleic acid sequence (SEQ ID NO:9) added to its 3' end) may comprise or consist of the nucleic acid sequence of SEQ ID NO:21 (see [link to invention]). Figure 23 ).

[0113] In this invention, the transposase expression vector comprising the nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 (which has an optimized C-myc nucleic acid sequence (SEQ ID NO:11) added to its 3' end) may comprise or consist of the nucleic acid sequence of SEQ ID NO:23 (see [link to invention]). Figure 23 ).

[0114] The present invention also provides an mRNA encoding a transposase represented by SEQ ID NO:12, which can be generated 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 containing a nucleic acid sequence encoding a transposase represented by SEQ ID NO:3.

[0115] The present invention also provides a transposase expressed by a transposase expression vector according to the invention or by mRNA encoding a transposase according to the invention, and according to one example of the invention, since the nucleic acid sequence encoding the transposase contained in the transposase expression vector or the template nucleic acid sequence for generating the mRNA encoding the transposase is optimized at the nucleic acid level, it contains the amino acid sequence represented by SEQ ID NO:13, which is identical to the amino acid sequence of existing transposase proteins.

[0116] In this invention, when the transposase expression vector contains a nucleic acid sequence encoding the transposase represented by SEQ ID NO:3, the transposase can be expressed by mRNA generated by in vitro transcription using the transposase expression vector, but is not limited thereto.

[0117] The present invention also provides a transposon system for delivering target DNA, comprising: a) A transposon vector containing inserted target DNA; and b) A transposase expression vector comprising a nucleic acid sequence encoding a transposase represented by SEQ ID NO:2 or SEQ ID NO:3, or an mRNA encoding a transposase represented by SEQ ID NO:12, or a transposase according to the invention.

[0118] In this invention, the term "transposon" refers to a polynucleotide capable of altering the position of a gene in the genome and integrating into a target site (e.g., the genomic or extrachromosomal DNA of a cell) by excision of a specific gene from a donor polynucleotide (e.g., a vector). A transposon is a polynucleotide comprising a nucleic acid sequence flanked by cis-acting nucleotide sequences, wherein 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 at each end of the transposon include at least one inverted repeat (IR), called an inverted terminal repeat (ITR), to which a transposase binds.

[0119] According to one example of the invention, the transposon vector and the 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, 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, but are not limited thereto.

[0120] Referring to Korean Patent No. 10-2602485, the transposon carrier of the present invention can be constructed according to conventional methods known in the art.

[0121] In this invention, the term "target DNA" refers to a foreign DNA molecule delivered to a cell using a transposon. The target DNA only needs to be able to insert into a transposon vector, be delivered to a target cell, and then be expressed. That is to say, it is clear that the target DNA is not limited to a specific type of DNA, and those skilled in the art can choose any desired target DNA without limitation according to its intended use.

[0122] In one embodiment of the present invention, the target DNA sequence may encode an antibiotic resistance protein, a therapeutic peptide, siRNA, miRNA, a reporter protein, a cytokine, a kinase, an antigen, an antigen-specific receptor, a cytokine receptor, a suicide peptide, a recombinant antibody, a neutralizing antibody against various viruses or other antigens, or a portion thereof, for example, may encode a chimeric antigen receptor (CAR), a T-cell receptor (TCR), or a portion thereof, but is not limited thereto.

[0123] In this invention, the term "therapeutic polypeptide" refers to a polypeptide or peptide that effectively prevents, improves, and / or treats any disease. Those skilled in the art may appropriately select polypeptides that exhibit therapeutic effects on a specific disease based on their intended use. The disease is not limited to any particular type; however, in one embodiment, the disease may be cancer.

[0124] The present invention also provides a transposon kit for delivering target DNA, comprising a transposon system for delivering target DNA according to the present invention and instructions.

[0125] The instruction manual may include a brochure, record, diagram, or other demonstration medium (e.g., CD, VCD, DVD, USB) for communication or to instruct on how to use the transposable subsystem of the present invention. The instruction manual may be attached to the container or may be packaged separately from the container containing the transposable subsystem of the present invention.

[0126] The kit may also include a container for containing the transposon subsystem of the present invention.

[0127] The kit may also include buffer solutions for stabilizing the transposon system and / or for performing cell transfection. Buffer solutions may be, for example, phosphate-buffered saline, Tris-based saline, Tris-EDTA buffer, piperazine ethanesulfonic acid buffer, or N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer, but are not limited to these.

[0128] The present invention also provides a cell in which: a) A transposon vector containing inserted target DNA; and b) A transposase expression vector containing a nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 or SEQ ID NO:3, or mRNA encoding the transposase represented by SEQ ID NO:12, or The transposase of the present invention.

[0129] In this invention, the cell can be a cell in which the target DNA is excised from the transposon vector by an intracellular transposase and integrated into the cell genome. That is, the target DNA can be inserted into the genome of the target cell using the transposon and transposase of this invention and expressed stably. The target DNA inserted into the cell genome can be expressed in the cell for 5 days or longer, 7 days or longer, 10 days or longer, 15 days or longer, 20 days or longer, or 30 days or longer after the transposon vector and transposase are introduced into the cell, but is not limited to these durations.

[0130] In other words, the present invention provides a transgenic cell in which target DNA is inserted into the genome via transposons. In this invention, the term "modification" refers to any manipulation of the cell that results in detectable changes, including but not limited to inserting heterologous or homologous polynucleotides and / or peptides into the cell, and mutating the cell's endogenous polynucleotides and / or peptides.

[0131] In one embodiment of the present invention, the cell may be one or more immune cells selected from a group consisting of myeloid cells such as T cells, B cells, natural killer (NK) cells, monocytes, macrophages, eosinophils, mast cells, basophils, granulocytes, and dendritic cells; or it may be stem cells derived from bone marrow, adipose tissue, peripheral blood, umbilical cord blood, or dental pulp, but is not limited thereto. Furthermore, the cell may be derived from insects, plants, fish, or mammals, particularly from human cells, but is not limited thereto.

[0132] According to one embodiment of the present invention, T cells can be activated by CD3 / CD28 beads, etc., before the introduction of transposon vectors, but are not limited thereto.

[0133] As used in this article, the term "immune cells" refers collectively to cells that play a role in the immune response.

[0134] Furthermore, after introducing transposon vectors and transposases (proteins or nucleic acid molecules), cells can be co-cultured with feeder cells. Feeder cells are support cells that do not proliferate themselves but provide extracellular secretions (such as growth factors) to facilitate the proliferation of cells in which target DNA has been introduced. Feeder cells are not limited to a specific type; any cell known in the art that can be used as a feeder cell can be used without limitation. Non-limiting examples include fibroblasts, human bone marrow mesenchymal cells, human amniotic epithelial cells, adipose-derived mesenchymal stem cells, and melanoma cells (A375 cells). Preferably, the feeder cells may be pre-irradiated before co-culturing with cells in which target DNA has been introduced.

[0135] When introducing CARs or TCRs into immune cells using the transposon system of this invention, co-culturing with feeder cells may particularly help improve the efficiency of gene delivery, as well as the proliferation and gene expression rate of the cells in which the gene has been introduced. For example, when the cells are T cells, they can be activated using TransAct or Dynabeads.

[0136] Co-culturing with feeder cells is preferred immediately after introducing the transposon vector and transposase into the cells via electroporation or the like, but is not limited to this. It 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 introduction.

[0137] The present invention also provides a method for inserting a target DNA sequence into a cell genome, the method comprising the steps of introducing a) and b) into the cell: a) A transposon vector containing inserted target DNA; and b) A transposase expression vector containing a nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 or SEQ ID NO:3, or mRNA encoding the transposase represented by SEQ ID NO:12, or The transposase of the present invention. The method may further include a step of co-culturing cells in which the transposon vector has been introduced with feeder cells after the introduction step.

[0138] As used herein, the term "introduction" refers to the delivery of a polynucleotide (e.g., a transposon vector or transposase vector) into a cell or organism. The nucleic acid of the polynucleotide can be in the form of naked DNA or RNA, can associate with a variety of proteins, or can be incorporated into a vector. The term "introduction" as used herein is intended to have the broadest possible meaning, including, for example, transfection methods (introducing polynucleotides into eukaryotic cells via physical and / or chemical treatment), transformation methods (introducing polynucleotides into prokaryotic cells via physical and / or chemical treatment), viral methods / viral transduction methods (introducing polynucleotides into eukaryotic and / or prokaryotic cells via viruses or viral vectors), conjugation methods (introducing polynucleotides from one cell to another via direct cell-to-cell contact or via a cytoplasmic bridge), and fusion methods (introduction via the fusion of two cells, including homo or heterocellular fusion). Preferably, this introduction is performed by electroporation.

[0139] The present invention also provides compositions for various uses, comprising, as an active ingredient, cells in which target DNA has been inserted into the genome via a transposon vector according to the invention. The cells may be autologous or allogeneic.

[0140] In one embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of immune-related diseases is provided, comprising the cells of the present invention as an active ingredient.

[0141] As used herein, the term "immune-related disease" refers to a disease and / or condition in which the immune system is involved in the pathogenesis of the disease, or where appropriate stimulation or suppression of the immune system may lead to the treatment and / or prevention of the disease. Examples of immune-related diseases that can be treated by this invention include, but are not limited to, tumors, infectious diseases, allergies, autoimmune diseases, graft-versus-host disease, or inflammatory diseases.

[0142] The inventors have demonstrated through specific examples that CAR T cells generated using the transposon of this invention differentiate into cytotoxic T cells and memory T cells in response to antigens. Therefore, those skilled in the art can generate transgenic cells with enhanced immune function by using the transposon of this invention to deliver appropriate antigen-specific CAR or TCR genes into immune cells, and these cells can be used to prevent or treat immune-related diseases.

[0143] For example, those skilled in the art can insert the gene encoding the target antigen into the transposon of the present invention and deliver it to immune cells to enhance the cellular immune function against the antigen. Enhanced immune function may mean, for example, activating the function of antigen-presenting cells, natural killer cells, and T cells (especially cytotoxic T cells) against the antigen, or regulating the activity of regulatory T cells, myeloid-derived suppressor cells (MDSCs), or M2 macrophages, but is not limited thereto.

[0144] Preferred embodiments are given below to aid in understanding the invention. However, the following embodiments are provided merely to facilitate a clearer understanding of the invention, and the scope of the invention is not limited to these embodiments. Example

[0145] Example A. Optimization of pBat transposase

[0146] To further improve the gene delivery efficiency of transposon systems, the DNA sequences of existing transposons were optimized using Genscript’s “Optimum GeneCodon Optimization” program to ensure their good expression in cells (especially T cells).

[0147] In addition, in order to improve the gene delivery efficiency of the transposon system by addressing the potential problems caused by DNA-type transposases, an mRNA-type transposase was prepared, which has low in vivo genotoxicity and a relatively short half-life (Example D).

[0148] [Table 1] Example B. Confirmation of gene delivery efficiency of pBat optimized transposase using optimized DNA sequence

[0149] To further improve the gene delivery efficiency of the pBat transposon system, the DNA sequence of the transposase was optimized to ensure good expression in cells, and the gene delivery efficiency was compared with that of existing transposases and the optimized transposase.

[0150] [method]

[0151] 1. DNA and cells

[0152] To confirm the gene delivery efficiency of the pBat optimized transposase with optimized DNA sequence, the pBat transposon 3M3-5M3-1G4 TCR (1.8 μg / μL) was used as the pBat transposon plasmid vector, and the pBat transposase plasmid (1.5 μg / μL) and the pBat optimized transposase plasmid (1.4 μg / μL) were used as pBat transposase plasmid vectors to express the transposase.

[0153] Peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals (LK045) and A375 cells irradiated with 50 Gy (ATCC, catalog number CRL-1619, batch number 70032966) were used as target cells.

[0154] 2. PBMC electroporation

[0155] Genes are transferred into cells via electroporation. The specific method is described below.

[0156] 1) Prepare 1.5 mL tubes according to the electroporation conditions shown in Table 2 below, and according to the conditions, measure 5.0 x 10⁻⁶ ppm. 6 Add 5 μg of transposase plasmid and transposon plasmid to each PBMC.

[0157] [Table 2] No. condition transposon plasmid transposase plasmid n 1 Electroporation (EP) only - - 2 2 Existing transposases 3M3-5M3-1G4 TCR Existing plasmids 2 3 Optimize transposases 3M3-5M3-1G4 TCR Optimize plasmids 2 2) 5.0 x 10 6 Each PBMC was added to a 1.5 mL tube of 1). 3) Take 5.0 x 10 from 2) 6 Carefully add 1 cell / 50 μL of cell suspension to the OC100 x 2 unit to avoid generating bubbles. 4) Select the resting T cell 14-3 protocol from MaxCyte STx for electroporation. 5) Insert the OC100 x 2 components from 3) into the STx and perform the electroporation procedure. 6) After electroporation, the cell suspension of the OC100 x 2 components (5.0 x 10) 6 (1 cell / 50μL / well) Transfer to flask T25. 7) Wash the OC100 x 2 components from 6) with 50 μL of AlyS medium and add it to each T25 flask. 8) After recovering the electroporated cells in a 37°C, 5% CO2 incubator for 20 minutes, add 2.0 x 10⁻⁶ cells to each flask. 6 Irradiated A375 cells were added to culture medium (ALyS + 3% HS + 200 mg / L). IU / mL IL-2) was added to make the total volume 3 mL. 9) Incubate in an incubator at 37°C and 5% CO2.

[0158] 3. T cell culture and storage after electroporation

[0159] 1) Three days after electroporation, add 5 mL of culture medium (ALyS + 3% HS + 200 IU / mL IL-2) and incubate in an incubator at 37°C and 5% CO2.

[0160] 2) Six days after electroporation, add 10 mL of culture medium (ALyS + 3% HS + 200 IU / mL IL-2) and incubate in an incubator at 37°C and 5% CO2.

[0161] 3) Seven days after electroporation, the cultured cells were resuspended, and 3 mL of suspension was collected from each flask for fluorescence-activated cell sorting (FACS) analysis.

[0162] 4) After 8 days of electroporation, the cells in suspension culture were transferred to new T25 flasks to divide them into two flasks. 8 mL of culture medium (ALyS + 3% HS + 200 IU / mL IL-2) was added to each flask and cultured in an incubator at 37°C and 5% CO2.

[0163] 5) Ten days after electroporation, collect 2 mL of suspension from each flask for FACS analysis. Transfer the remaining cells from the T25 flask to each T75 flask, add 10 mL of culture medium (ALyS + 3% HS + 200 IU / mL IL-2) to each flask, and incubate at 37°C in a 5% CO2 incubator.

[0164] 6) After 13 days of electroporation, add 10 mL of culture medium (ALyS + 3% HS + 200 IU / mL IL-2) and incubate in an incubator at 37°C and 5% CO2.

[0165] 7) After 14 days of electroporation, the cultured cells were resuspended, and 2 mL of suspension was collected from each flask for FACS analysis.

[0166] 8) After 14 days of electroporation, the remaining cells in the flask from step 7) were transferred to each 50 mL conical tube.

[0167] 9) Centrifuge at 1,500 rpm for 5 minutes at room temperature and remove the supernatant.

[0168] 10) Suspend the cell clumps in 5 mL of ALyS medium and count the cells.

[0169] 11) Centrifuge at 1,500 rpm for 5 minutes at room temperature and remove the supernatant.

[0170] 12) Suspend the cell clumps in 1 mL of CS10, freeze the suspension using a controlled rate freezer (CRF) (ThermoFisher, CryoMed TSCM34PV, S / N 300503011, 300503015) according to Protocol 6, and store in a nitrogen tank.

[0171] 4. FACS Analysis

[0172] FACS analysis was performed at 7, 10 and 14 days after electroporation.

[0173] 1) Suspend the cells in each group of flasks and transfer them to FACS tubes, 2 to 3 mL per tube.

[0174] 2) Centrifuge at 4°C and 1,500 rpm for 5 minutes, remove the supernatant, and resuspend the cells in 1 mL of FACS buffer (phosphate-buffered saline (PBS) + 2% fetal bovine serum (FBS)).

[0175] 3) Repeat step 2) to wash again.

[0176] 4) Centrifuge at 4°C and 1,500 rpm for 5 minutes to remove the supernatant.

[0177] 5) Add 5 μL of human TruStain FcX + 95 μL of FACS buffer to each tube and incubate at room temperature for 5 minutes.

[0178] 6) Add 2 μL of each antibody (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, anti-CCR7, anti-mTCRβ) to each tube and incubate in the dark at room temperature for 30 minutes.

[0179] 7) Add 1 mL of FACS buffer (PBS + 2% FBS) to each tube and centrifuge at 4°C and 1,500 rpm for 5 minutes.

[0180] 8) Remove the supernatant and then resuspend the cells in 1 mL of FACS buffer (PBS + 2% FBS).

[0181] 9) Centrifuge at 4°C and 1,500 rpm for 5 minutes to remove the supernatant.

[0182] 10) Cells were resuspended in 200 μL FACS buffer (PBS + 2% FBS), with or without 1 x 4',6-diamidinyl-2-phenylindole (DAPI), and analyzed by FACS.

[0183] [result]

[0184] 1. Analysis of 1G4 TCR expression 7 days after electroporation

[0185] Since the constant region of the 1G4 TCR consists of a fixed gene sequence of the mouse TCR, 1G4 TCR expression was confirmed by using an anti-mTCRβ antibody through the expression of mTCRβ. Figure 1 As shown, the results of confirming the proportion of CD3+ T cells expressing 1G4TCR 7 days after electroporation were as follows: in the "EP-only group (negative control group)" where electroporation was performed without plasmids, the proportion was 0%, while in the 3M3-5M3-1G4 TCR transposon, the proportion was 27.7% in the "existing transposon group" and 56.0% in the "optimized transposon group". This confirms that the proportion of mTCRβ-expressing cells is twice as high when using optimized transposases.

[0186] 2. Analysis of 1G4 TCR expression 10 days after electroporation

[0187] As a result confirming the proportion of CD3+ T cells expressing 1G4 TCR 10 days after electroporation, such as Figure 2 As shown, in the "EP only group (negative control group)", this proportion was 0%, and in the transposon group, this proportion was generally increased compared to 7 days after electroporation. Specifically, in the 3M3-5M3-1G4 TCR transposon, this proportion was 62.7% in the "existing transposession group" and 79.9% in the "optimized transposession group". Therefore, similar to 7 days after electroporation, the proportion of mTCRβ-expressing cells was 1.3 times higher when using the optimized transposession.

[0188] 3. Analysis of 1G4 TCR expression 14 days after electroporation

[0189] As a result confirming the proportion of CD3+ T cells expressing 1G4 TCR 14 days after electroporation, such as Figure 3 As shown, the proportion was 0% in the "EP only group (negative control group)" and generally similar to that at 10 days post-electroporation in the transposon group. Specifically, in the 3M3-5M3-1G4TCR transposon, the proportion was 59.0% in the "existing transposon group" and 76.7% in the "optimized transposon group." Therefore, similar to 7 and 10 days post-electroporation, the proportion of mTCRβ-expressing cells was 1.3 times higher when using the optimized transposon.

[0190] Furthermore, when comparing the proportion of 1G4 TCR-expressing T cells at 7, 10, and 14 days after electroporation, such as Figure 4As shown, the proportion of 1G4 TCR-expressing T cells increased at 10 and 14 days compared to 7 days. In the comparison of transposases, at 7, 10, and 14 days after electroporation, the proportion of 1G4 TCR-expressing cells in the "optimized transposase" was higher than that in the "existing transposase," indicating that the optimized transposase has better gene delivery efficiency.

[0191] 4.1 Analysis of G4 TCR expression in CD4+ and CD8+ cells in T cells

[0192] The proportions of helper T cells (CD4+ T cells) and cytotoxic T cells (CD8+ T cells) in 1G4 TCR-expressing T cells were compared. In the "EP only group," CD4+ and CD8+ cells were confirmed in CD3+ T cells, but no 1G4 TCR-expressing T cells were found. The results are as follows: Figure 5 As shown, after 7 days, the proportions of CD4+ and CD8+ cells in the "EP only group" were similar, while after 10 days, CD4+ cells accounted for 33.0% and CD8+ cells for 62.5%, indicating a higher abundance of CD8+ cells. After 14 days, CD4+ cells accounted for 23% and CD8+ cells for 72.5%, indicating a higher abundance of CD8+ cells. This may be because the proportion of CD8+ cells among CD3+ T cells increases with the proliferation of CD8+ cells during culture.

[0193] Furthermore, as a result confirming the presence of CD4+ and CD8+ cells in CD3+mTCRβ+ T cells within the 3M3-5M3-1G4 TCR transposon, such as Figure 5 and Figure 6 As shown, in the "existing transposomyome," the proportion of CD4+ cells was 40.5% after 7 days, 44.9% after 10 days, and 29.0% after 14 days; the proportion of CD8+ cells was 55.5% after 7 days, 51.8% after 10 days, and 67.2% after 14 days. It was confirmed that the difference between CD4+ and CD8+ cells was small within 10 days, but after 14 days, the proportion of CD8+ cells increased 2.3-fold. On the other hand, in the "optimized transposomyome," the proportion of CD4+ cells was 24.7% after 7 days, 22.1% after 10 days, and 14.4% after 14 days; the proportion of CD8+ cells was 71.4% after 7 days, 74.8% after 10 days, and 82.2% after 14 days. Therefore, it was confirmed that CD8+ cells gradually increased to 2.9 times that of CD4+ cells after 7 days, 3.4 times after 10 days, and 5.7 times after 14 days.

[0194] 5.1 Analysis of memory T cells in T cells expressing G4 TCR

[0195] In 1G4 TCR-expressing T cells, the proportion of memory T cells was analyzed using CD45RA and CCR7 markers. When using CD45RA and CCR7 markers to distinguish memory T cells, CD45RA+CCR7- T cells were classified as T cells. EFF Cells, CD45RA-CCR7-T cells are classified as T cells. EM Cells, CD45A-CCR7+ T cells are classified as T cells. CM Cells, CD45VA+CCR7+ T cells were classified as T cells. SCM Cells. In the "EP only group," memory T cells were identified in CD3+ T cells because no 1G4 TCR-expressing T cells were present. Results, as... Figure 7 As shown, this confirms that T increases with increasing culture time. CM and T SCM Reduce T EM Increase. For example, Figure 7 As shown in Table 3, the proportion of memory T cells in 1G4 TCR-expressing T cells within the "existing transposase genome" is as follows: After 7 days, for T... EFF It was 29.7%, for T EM It is 35.3%, for T CM It was 15.1%, and for T SCM It was 20.0%; 10 days later, for T EFF It was 39.8%, for T EM It is 31.0%, for T CM It was 14.2%, and for T SCM It was 15.1%; 14 days later, for T EFF It is 38.3%, for T EM It is 38.8%, for T CM It was 8.2%, and for T SCM The figure was 14.8%, confirming T. CM and T SCM Gradually decreasing, while T EM and T EFF Gradually increase. In "Optimizing Transposomies," the ratio is as follows: After 7 days, for T... EFF It was 29.7%, for T EM It is 32.8%, for T CM It was 14.4%, and for T SCM It was 23.1%; 10 days later, for T EF F is 42.8%, for T EM It is 19.5%, for T CM It was 14.4%, and for T SCM It was 22.5%; and 14 days later, for T EFFIt is 38.7%, for T EM It is 26.1%, for T CM It was 8.4%, and for T SCM The figure was 27.0%, confirming T. CM Reduce T SCM Increase, T CM and T SCM The combined cell ratio is higher than that of existing transposases.

[0196] [Table 3]

[0197] As described above, when producing 1G4 TCR-T cells using the pBat transposon system in PBMCs, it was confirmed whether there was a difference in the delivery and expression efficiency of the 1G4 TCR gene based on the use of existing or optimized transposases. The results confirmed that the optimized transposase had higher gene delivery efficiency in LK045 PBMCs. Furthermore, regarding the proportion of CD4+ or CD8+ cells in 1G4 TCR-expressing cells, when using the existing transposase, the proportion of CD8+ T cells was 2.3 times that of CD4+ T cells after 14 days, but when using the optimized transposase, the proportion of CD8+ T cells was 2.9 times after 7 days, and the difference gradually increased thereafter. As a result of confirming memory type using CD45RA and CCR7 markers in 1G4 TCR-expressing T cells, it was confirmed that the optimized transposase contained T... CM and T SCM The proportion of cells is very high. Therefore, when using the pBat transposon system to produce TCR-T cells, and when using optimized transposases, TCR-T cells with a high proportion of cytotoxic T (CD8+T) cells can be produced in high yield.

[0198] Example C. Confirmation of optimized transposase gene delivery efficiency in Jurkat cells

[0199] 1. Method

[0200] Jurkat cells (ATCC, catalog number TIB-152, batch number 70017560) were used as target cells to confirm the optimized gene delivery efficiency of transposases.

[0201] The pBat transposon 3M3-5M3-EGFP vector containing EGFP was used as the transposon plasmid vector, and the transposase delivery efficiency was compared using the pBat transposase plasmid and the pBat optimized transposase plasmid.

[0202] The fluorescence of green fluorescent protein (GFP) 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.

[0203] 2. Results

[0204] On the first day, such as Figure 8a and Figure 8b As shown, there was no difference in the proportion of GFP-expressing cells between the two transposases. On day 7, as... Figure 9a and Figure 9b As shown, there was no difference in the proportion of cells expressing total GFP between the two types of transposases, but in the optimized transposase, the proportion of cells expressing high GFP was confirmed to be approximately twice as high. On day 14, as... Figure 10a and Figure 10b As shown, in the optimized transposase, the ratio of cells expressing total GFP to cells expressing high levels of GFP was confirmed to be approximately twice as high.

[0205] The results confirm that the GFP expression rate of the optimized transposase is twice that of existing transposases.

[0206] Example D. Confirmation of improved transposase gene expression efficiency by optimizing mRNA-type pBat

[0207] The results of comparing the gene delivery efficiency of existing transposases and codon-optimized transposases confirmed that codon-optimized transposases exhibit higher gene delivery efficiency.

[0208] However, when excessive amounts of DNA-type transposases are used for gene delivery, in vivo toxicity is induced due to intracellular mechanisms that recognize foreign DNA. Furthermore, the inserted foreign gene may be remobilized when the transposase gene delivered to the cell in DNA form continues to exist in the cell and transposase expression persists for a long time. Therefore, an mRNA-type transposase with low in vivo toxicity and a relatively short half-life was developed, and its gene delivery efficiency was compared with that of the DNA-type transposase.

[0209] [method]

[0210] 1. Cells and DNA

[0211] Jurkat, clone E6-1 (ATCC, catalog number TIB-152, batch number 70017560), and PBMCLK053 isolated from healthy individuals were used as target cells to confirm the increased gene expression efficiency of mRNA-type pBat transposases.

[0212] pBat transposon vectors containing the GFP gene and pBat transposon vectors containing the 1G4 TCR gene were used as transposon plasmid vectors. In order to confirm the gene delivery efficiency based on the delivery mode of the transposase gene, plasmid DNA-type optimized transposase (pBat-optimized transposase plasmid) and mRNA-type optimized transposase were used.

[0213] 2. Production of template DNA for mRNA synthesis

[0214] The optimization of the existing transposase gene sequence (SEQ ID NO:1) to adapt it to mRNA form was entrusted to GenScript CO.,LTD., and the synthesis of the DNA sequence (SEQ ID NO:3) optimized for mRNA form in the GenSmart optimization report (tool version Beta 1.0) was entrusted to Bionics CO.,LTD. The gene of the DNA sequence optimized for mRNA form was cloned into TaKaRa IVTpro. TM The cloning kit for the mRNA template (Cat.#6143) in the T7 mRNA synthesis kit is as follows.

[0215] For infused clones with DNA sequences optimized for mRNA, PCR insertion amplification was performed using a DNA sequence (SEQ ID NO:3) synthesized by Bionics CO.,LTD. as a template, and the PCR product was loaded onto an agarose gel. Figure 11 As shown, confirm the desired band size, and then perform gel extraction. In Takara IVTpro... TM The T7 mRNA synthesis system includes a cloning kit for mRNA templates (vectors) and an infusion cloning process between a DNA sequence (insertion, SEQ ID NO:3) optimized for mRNA form, and as... Figure 12 As shown, the structure was confirmed by enzyme mapping to prepare the pBat optimized transposase mRNA template plasmid required for the production of the optimized transposase in mRNA form using an in vitro transcription method.

[0216] 3. Optimize the in vitro transcription (IVT) process by converting pBat transposases into mRNA.

[0217] To synthesize mRNA using the pBat template plasmid produced in “2.” above, the template plasmid was linearized with Hind III restriction enzyme and subjected to an IVT reaction as described below.

[0218] 1) Add the restriction enzyme Hind III to the pBat optimized transposase mRNA template plasmid and linearize it by reacting at 37°C for 3 hours.

[0219] 2) The linearized mRNA template plasmid was purified using EtOH precipitation, as described below:

[0220] 2-1) Add 20 μL of 3M sodium acetate to the reaction product in 1) and mix thoroughly. Cool the resulting mixture at -20°C for more than 15 minutes.

[0221] 2-2) Centrifuge at 12,000 rpm and 4°C for 15 minutes.

[0222] 2-3) Carefully discard the supernatant, add 1 mL of 70% EtOH, and centrifuge at 12,000 rpm and 4°C for 15 minutes.

[0223] 2-4) Carefully discard the supernatant and thoroughly dry the DNA clumps.

[0224] 2-5) Add 200 μL of nuclease-free water to bring the final concentration to 0.5 to 1.0 μg / μL, and store the resulting mixture at -20°C until use.

[0225] 3) Put Takara IVTpro TM Dissolve and mix the reagents included in the T7 mRNA synthesis system at room temperature, then spun down. Place the 10X enzyme mixture on ice.

[0226] 4) Add the reagents to tube E in the order shown in Table 4 below. The total reaction volume is 120 μL.

[0227] [Table 4]

[0228] 5) After thorough mixing, react at 37°C for two hours. Then, treat 24 μL of LDNase I in tube E containing the reaction product from 4) and react at 37°C for 15 minutes.

[0229] 6) As described below, the mRNA-type optimized transposase was purified by LiCl precipitation.

[0230] 6-1) Add 180 μL of nuclease-free water to 5), and add 180 μL of LiCl.

[0231] 6-2) After thorough mixing, allow the resulting mixture to react at -20°C for at least 30 minutes, and then centrifuge at 12,000 rpm and 4°C for 15 minutes.

[0232] 6-3) Carefully discard the supernatant, add 1 mL of 70% EtOH, and centrifuge at 12,000 rpm and 4°C for 15 minutes.

[0233] 6-4) Carefully discard the supernatant, thoroughly dry the DNA clumps, and dissolve the DNA in 200 μL of nuclease-free water.

[0234] 6-5) Take 10 μL of each sample and store at -80℃ until used.

[0235] 4. PBMC Activation

[0236] Gene delivery efficiencies for two different genes (GFP / 1G4 TCR) in PBMCs were compared based on transposon type using the Neon and Maxyte electroporation devices (see Table 5). When delivering the GFP gene to PBMCs using a transposon system equipped with the Neon device, Miltenyi's TransAct was used. TM PBMCs were activated for three days without the addition of feeder cells during cell culture. On the other hand, when the 1G4 TCR gene was delivered to PBMCs using a transposon system with a MaxCyte device, the PBMCs were not activated, but feeder cells were added during cell culture.

[0237] [Table 5] Electroporation equipment Gene expression PBMC activation Feeder cells Neon GFP conduct No addition MaxCyte 1G4 TCR No Add to

[0238] 1) Prepare Roswell Park Memorial Institute (RPMI) medium (RPMI + 10% FBS + 1X P / S) by preheating in a 37°C water bath.

[0239] 2) Remove PBMC LK053 from the nitrogen tank and thaw it rapidly in a 37°C water bath.

[0240] 3) Prepare 30 mL of culture medium in a 50 mL tube and add thawed PBMC.

[0241] 4) Centrifuge at 1,500 rpm for 5 minutes, remove the supernatant, suspend the cells in 40 mL of RMPI medium, and count the cells.

[0242] 5) 1.2 x 10 8 One PBMC was placed in a T175 flask, and RPMI medium was added to prepare a solution of 1.2 x 10⁻⁶ PBMCs. 8 / 120mL.

[0243] 6) Add IL-2 to a final concentration of 20 IU / mL, every 2.0 x 10 7 Add 100 μL of Transact (IL-2) to each cell at a concentration of 1.0 x 10⁻⁶. 6 Dilute the IU / mL culture medium by 1 / 10 to prepare 1.0 x 10⁻⁶ liters. 5 (IU / mL).

[0244] 7) Incubate at 37°C in a 5% CO2 incubator for two days.

[0245] 5. PBMC electroporation

[0246] 5-1. Neon device conditions (PBMC activation; no feeder cells added)

[0247] 1) Dispense 3 mL of E2 buffer stored at 4 °C into each of three Neon tubes.

[0248] 2) Inoculate 1.4 mL of culture medium (AlyS + 3% HS + IL-2 200 IU / mL) into each well of a 12-well plate. At this point, four wells are prepared for each group.

[0249] 3) Harvest fully activated PBMC cells.

[0250] 4) Centrifuge at 1,500 rpm for 5 minutes at room temperature.

[0251] 5) After removing the supernatant, suspend the cell clumps in 1.0 mL of Opti-MEM buffer.

[0252] 6) Centrifuge at 1,500 rpm for 5 minutes at room temperature.

[0253] 7) After removing the supernatant, resuspend the cell clumps in Opti-MEM buffer to 1.0 x 10⁻⁶. 6 / 100μL.

[0254] 8) For each condition, the amount of transposon and transposase plasmid added is 1.0 x 10⁻⁶. 6 3 μg of each cell was added, as shown in Table 6 below (no vector was added in the control group). The mixture of cells and plasmids was then thoroughly mixed by pipetting.

[0255] [Table 6]

[0256] 9) Install the Neon tube containing the E2 buffer from 1) onto the Neon device.

[0257] 10) Using the Neon pipette and Neon tip, slowly aspirate the 100 UI cell suspension from 8) and insert it into the Neon device.

[0258] 11) After electroporation at 1,700 V, 20 ms and 1 pulse, the completed PBMCs were inoculated into each well of a 12-well plate containing culture medium and incubated in an incubator at 37 °C and 5% CO2.

[0259] 12) One day after transfection, cells were harvested from two wells (observation plate after 1 day) under each condition, and FACS analysis was performed. 2 mL of culture medium was added to each remaining second well (observation plate after 7 days). The resulting mixture was transferred to a T75 flask and cultured in an incubator at 37°C and 5% CO2.

[0260] 13) Five days after transfection, add 2 mL of culture medium and incubate in an incubator at 37°C and 5% CO2.

[0261] 14) Seven days after transfection, PBMCs were resuspended by pipetting the culture medium from each flask for FACS analysis, and 1 mL of cells was collected from a total of 4 mL. 5 mL of culture medium was added to the remaining culture medium, and then the cells were cultured.

[0262] 15) On days 8, 11 and 13 after transfection, 5 mL, 10 mL and 20 mL of fresh culture medium were added respectively, and the culture was carried out in an incubator at 37°C and 5% CO2.

[0263] 16) Fourteen days after transfection, PBMCs were resuspended by pipetting the culture medium from each flask for FACS analysis, and cells were collected in a volume of 200 UI.

[0264] 5-2. MaxCyte equipment conditions (PBMCs not activated; feeder cells added)

[0265] 1) Remove PBMC LK053 from the nitrogen tank, thaw it rapidly in a 37°C water bath, then prepare 30 mL of culture medium in a 50 mL tube and add PBMC.

[0266] 2) Centrifuge at 1,500 rpm for 5 minutes to remove the supernatant.

[0267] 3) Suspend the cell clumps in 20 mL of culture medium (ALYS505N-O+3% HS) and count the cells.

[0268] 4) Centrifuge at 1,500 rpm for 5 minutes at room temperature.

[0269] 5) Remove the supernatant and suspend the cell clumps in 20 mL of Dulbecco's phosphate buffered saline (DPBS).

[0270] 6) Centrifuge at 1,500 rpm for 5 minutes at room temperature to completely remove the supernatant, and suspend the cell clumps in 5 mL of Opti-MEM medium.

[0271] 7) Centrifuge at 1,500 rpm for 5 minutes at room temperature to completely remove the supernatant.

[0272] 8) Suspend the cell clumps in 50 μL of warm Opti-MEM medium to prepare 5.0 x 10⁻⁶ cells / mL. 6 / 50μL.

[0273] 9) As shown in Table 7 below, the transposon vector and transposase vector were added to the tubes in 8) at a rate of 5 μg per well (no vector was added to the control group).

[0274] [Table 7] No. Group Cell count transposus Optimize transposase types 1 Electroporation <![CDATA[5.0x 10 6 Individual cells]]> - - 2 pBat-1G4 TCR <![CDATA[5.0x 10 6 Individual cells]]> pBat 3M3-5M3 plasmid DNA type 3 pBat-1G4 TCR <![CDATA[5.0x 10 6 Individual cells]]> pBat 3M3-5M3 mRNA type

[0275] 10) will come from 5.0x 10 of 9). 6 One cell / 50 μL cell suspension was carefully added to each OC100X2 unit to avoid generating bubbles.

[0276] 11) Select the resting T cell 14-3 protocol for electroporation in MaxCyte STx.

[0277] 12) Insert the OC100x2 assembly from 10) into the chamber of the STx and perform an electroporation procedure.

[0278] 13) After electroporation, transfer the cell suspension from the OC100x2 assembly to a T25 flask (5.0 x 10⁻⁶). 6 (1 cell / 50μL / well).

[0279] 14) Wash the OC100X2 wells with 50 μL of Opti-MEM medium and add the cell suspension from 13) to each well of the plate.

[0280] 15) Provide a 20-minute recovery time in a 37°C, 5% CO2 incubator.

[0281] 16) Thaw the A375 cell stock solution irradiated with 100 Gy rapidly in a 37°C water bath, slowly add it to 10 mL of culture medium, and centrifuge at 1,500 rpm for 5 minutes.

[0282] 17) Remove the supernatant, suspend the cells in 10 mL of culture medium, and count the cells.

[0283] 18) Centrifuge at 1,500 rpm for 5 minutes at room temperature to completely remove the supernatant.

[0284] 19) Resuspend the cells in complete culture medium (ALYS505N-O + 3% HS + 200 IU / mL IL-2) to a final volume of 2.0 x 10⁻⁶. 6 1 cell / 3mL.

[0285] 20) After completing the process in 15), add 3 mL of the cell suspension in 19) to each flask, and then incubate for two days in a 37°C, 5% CO2 incubator.

[0286] 21) Two days after electroporation, add 2 mL of culture medium and IL-2 to each flask to a final concentration of 200 IU / mL, and incubate at 37°C in a 5% CO2 incubator.

[0287] 22) Five days after electroporation, add 10 mL of culture medium and IL-2 to a final concentration of 200 IU / mL, and incubate at 37°C in a 5% CO2 incubator.

[0288] 23) Seven days after electroporation, the cultured cells were resuspended, and 500 μL of the cell suspension was transferred to a new tube for FACS analysis. The remaining cell suspension was adjusted to a total of 15 mL, and new culture 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.

[0289] 24) Nine days after electroporation, add 5 mL of culture medium (ALYS505N-O+3% HS) to make a total volume of 20 mL, add IL-2 to a final concentration of 200 IU / mL, and incubate at 37°C in a 5% CO2 incubator.

[0290] 25) Twelve days after electroporation, add 5 mL of culture medium (ALYS505N-O+3% HS) to make a total volume of 25 mL, add IL-2 to a final concentration of 200 IU / mL, and incubate in an incubator at 37°C and 5% CO2.

[0291] 26) Fourteen days after electroporation, the cultured cells were resuspended, and 500 μL of the cell suspension was transferred to new tubes for FACS analysis. Two x 10⁻⁶ cells were harvested from each group. 7 Cells were subjected to in vitro killing assays and cultured in 20 mL of resting medium (ALYS505N-O + 3% HS) for 24 hours. After removing the supernatant, CS10 medium was added, and the resulting mixture was stored at -80°C for 1 day. Then, it was transferred to a nitrogen tank, and the remaining suspension was used to prepare the stock solution.

[0292] 6. FACS Analysis

[0293] FACS analysis was performed 7 and 14 days after electroporation.

[0294] 1) After suspending the cells in each group of flasks, take 500 μL and transfer it to FACS tubes.

[0295] 2) Centrifuge at 4°C and 1,500 rpm for 5 minutes, remove the supernatant, and resuspend the cells in 1 mL of washing buffer (PBS + 2% FBS).

[0296] 3) Repeat step 2) to wash again.

[0297] 4) Centrifuge at 4°C and 1,500 rpm for 5 minutes to remove the supernatant.

[0298] 5) Add 5 μL of human TruStain FcX + 95 μL of FACS buffer to each tube and incubate at room temperature for 5 minutes.

[0299] 6) Add 2 μL of each antibody (anti-CD3, anti-CD4, anti-CD8, anti-CD45RO, anti-CD62L) to each tube, and add 0.5 μL of anti-mTCRβ antibody to each tube. Incubate the resulting mixture in the dark at room temperature for 30 minutes.

[0300] 7) Add 1 mL of FACS buffer (PBS + 2% FBS) and centrifuge at 4°C and 1,500 rpm for 5 minutes.

[0301] 8) Remove the supernatant and resuspend the cells in 1 mL of FACS buffer (PBS + 2% FBS).

[0302] 9) Centrifuge at 4°C and 1,500 rpm for 5 minutes to remove the supernatant.

[0303] 10) Resuspend cells in 200 μL of FACS buffer (PBS + 2% FBS) containing 1x DAPI and perform FACS analysis.

[0304] 7. Peptide pulses in T2-Luc cells

[0305] 1) T2-Luc cells cultured in Iscove modified DuPont medium (IMDM) (IMDM + 10% FBS + 1X P / S) were collected in 15 mL conical tubes.

[0306] 2) Centrifuge at 1,500 rpm for 5 minutes at room temperature.

[0307] 3) Remove the supernatant, suspend the cell clumps in 5 mL of IMDM, and count the cells.

[0308] 4) Add IMDM to prepare 1.0 x 10⁻⁶ 6 / mL, and place the resulting mixture into a polypropylene (PP) tube according to the conditions shown in Table 8 below.

[0309] [Table 8] Tube condition T2 cell count β2m treatment concentration Peptide treatment concentration 1 No pulse <![CDATA[1.0x 10 6 ]]> - - 2 NY-ESO-1 pulse <![CDATA[1.0x 10 6 ]]> 3μg / mL 100 μg / mL

[0310] 5) Add 100 mg / mL NY-ESO-1 peptide and 0.5 mg / mL β2m to the T2-Luc cell pulse group to achieve the same treatment concentration as the peptide "pulse" condition in Table 8 above. Add the same volume of dimethyl sulfoxide (DMSO) as the added peptide to the "no pulse" group.

[0311] 6) The reaction is carried out for two hours, and tapped once every 30 minutes at room temperature.

[0312] 7) Centrifuge at 1,500 rpm for 5 minutes at room temperature and remove the supernatant.

[0313] 8) Resuspend the cell clumps in 10 mL of PBS and centrifuge at 1,500 rpm for 5 minutes at room temperature.

[0314] 9) Remove the supernatant, suspend the cell clumps in 1 mL of ALyS medium (ALYS505N-O+3% HS), and then count the cells.

[0315] 10) By mixing ALYS medium (ALYS505N-O+3% HS) at 2.0 x 10⁻⁶ ppm... 4 Target cells were prepared by adding a concentration of 50 μL to T2-Luc cells.

[0316] 8.1 Cytotoxicity assay based on luciferase after co-culture of G4 TCR-T cells and pulsed T2-Luc cells

[0317] 1) 1G4 TCR-T cells that have been resting for 24 hours under '5-2.MaxCyte equipment conditions' were collected in 50 mL conical tubes.

[0318] 2) Centrifuge at 1,500 rpm for 5 minutes at room temperature.

[0319] 3) Remove the supernatant, suspend the cell clumps in 10 mL of ALyS medium (ALYS505N-O+3% HS), and count the cells.

[0320] 4) Add ALyS medium (ALYS505N-O + 3% HS) to achieve a 1G4 TCR-T cell concentration of 6.0 x 10⁻⁶. 5 / 50μL, to prepare effector cells.

[0321] 5) To achieve effector cell (E) to target cell (T) ratios of 30:1, 10:1, 3:1, and 1:1 in two 96-well white plates, first, seed 50 μL of culture medium into channels B to D and F to H according to the conditions described in the table below, and then seed 9.0 x 10⁻⁶ cells / well of the target cell culture medium. 5 75 μL of 1G4 TCR-T cells (which are effector cells (E)) were added to channels A and E. Serial dilutions of 25 μL were then performed in channels A through D and E through H using multichannel pipettes.

[0322] 6) Add 2.0 x 10 according to the conditions in the table below. 4 (50 μL) T2-Luc cells prepared in “7” above, and mix thoroughly.

[0323] 7) Add only T2 cells according to the conditions in Tables 9 and 10, and add 20 μL of 10% Triton-X to some “T2 only (Lysis)” wells and mix thoroughly.

[0324] 8) Adjust the total volume of all wells to 100 μL using ALyS medium.

[0325] [Table 9] [Table 10]

[0326] 9) Co-culture for 4 hours in a 37℃, 5% CO2 incubator.

[0327] 10) Add 100 μL of luciferase assay reagent to each well, cover the well with aluminum foil, and allow the mixture to react at room temperature for two minutes.

[0328] 11) Place the 96-well plate in a spectrophotometer and measure the luminescence.

[0329] [result]

[0330] 1. Cloning of mRNA transposases

[0331] For infusion clones, PCR was performed using a DNA sequence (SEQ ID NO:3) optimized for the mRNA form synthesized by Bionics CO.,LTD. as a template for insertion amplification. After loading onto an agarose gel, as shown... Figure 11 The desired band size is confirmed as shown, and then gel extraction is performed.

[0332] Subsequently, at Takara IVTpro TMThe T7 mRNA synthesis system includes a cloning kit for the mRNA template (vector) and an infusion cloning process between the DNA sequence (insertion, SEQ ID NO. 3) optimized for mRNA form. After extracting the cloned plasmid DNA, the final candidate clones are selected by enzyme mapping treated with Cla I and Nde I restriction enzymes, with structures as shown below. Figure 12 As shown.

[0333] 2. Confirmation of total cell count in culture

[0334] After delivering the 1G4 TCR gene to PBMCs via MaxCyte electroporation, the total cell count and cell viability were determined by culturing the cells with feeder cells for 14 days. Regarding the total cell count, as... Figure 13 As shown, the proliferation rate was confirmed to vary slightly depending on the delivery pattern of the transposase gene. The initial cell number at electroporation was 5.0 x 10⁻⁶. 6 The cell count was 1.3 x 10^12 cells, but after 14 days of culture, the group that did not undergo electroporation proliferated to 1.3 x 10^12 cells. 7 The highest proliferation rate observed in the group using DNA-optimized transposases was 1.4 x 10⁻⁶ cells. 7 Cells using the mRNA-optimized transposase group showed the highest proliferation rate of 1.8 x 10⁻⁶. 7 Cells. In addition, such as Figure 14 As shown, cell viability was confirmed to be 89.4% in the group without electroporation, 91.7% in the group using DNA-optimized transposase, and 90.3% in the group using mRNA-optimized transposase.

[0335] 3. FACS analysis was performed based on the optimized transposase gene delivery type after Neon electroporation (CD3 / CD28 bead activation; no feeder cells added).

[0336] To confirm GFP gene expression based on the mRNA and DNA forms of the transposase gene in PBMCs, gene expression was confirmed by FACS analysis on days 7 and 14 after transformation by Neon electroporation. In this experiment, PBMCs were activated with CD3 / CD28 beads prior to electroporation and cultured without feeder cells. Figure 15 As shown, the efficiency and expression of GFP gene delivery to CD3+ T cells were confirmed by gating in the order of lymphocytes, monomers, live cells, CD3+ T cells and GFP+ T cells.

[0337] To confirm GFP gene expression based on mRNA-optimized and DNA-optimized transposases in PBMC cells, the proportion of CD3+ T cells expressing GFP was determined on day 7 of culture. Results were as follows: Figure 16As shown, after 7 days of culture, GFP was confirmed to be absent in the negative control group (EP only) undergoing electroporation, and the proportions in the transposon-only group (pBat 5098 (pBat 3M3-5M3)) were 0.06% and 0.02%, indicating almost no GFP expression. On the other hand, in the DNA-optimized transposesase group, GFP expression rates were 8.5% and 7.4%, while in the mRNA-optimized transposesase group they were 18.2% and 18.5%, confirming that the GFP expression rate in the mRNA-optimized transposesase group was approximately 10% or more higher. Furthermore, after 14 days of culture, the GFP expression rates in the DNA-optimized transposesase group were 3.0% and 3.8%, while the GFP expression rates in the mRNA-optimized transposesase group were 8.3% and 8.8%, confirming a similar result to that obtained on day 7, with the mRNA-optimized transposesase group showing an expression rate approximately 5% or more higher.

[0338] 4. FACS analysis was performed based on the optimized transposase gene delivery type after MaxCyte electroporation (unactivated; feeder cells added).

[0339] To confirm the expression of the 1G4 TCR gene in PBMC cells based on the mRNA and DNA forms of the transposase gene, FACS analysis was performed on days 7 and 14 after transformation by MaxCyte electroporation. In this experiment, PBMCs were not activated with CD3 / CD28 beads before electroporation; instead, they were cultured with feeder cells. Regarding the analytical methods, see below. Figure 17 As shown, the efficiency and expression of the 1G4 TCR gene in CD3+ T cells were confirmed by gating in the order of lymphocytes, monomers, live cells, CD3+ T cells, and 1G4+ T cells.

[0340] 4-1. Comparison of 1G4 TCR gene expression based on optimized transposase gene delivery type

[0341] To confirm the expression of the 1G4 TCR gene based on mRNA-optimized and DNA-optimized transposases in PBMC cells, the proportion of CD3+ T cells expressing 1G4 TCR was determined on day 7 of culture. The results are as follows: Figure 18As shown, after 7 days of culture, it was confirmed that 1G4 TCR was not expressed in the negative control group "EP only," which only underwent electroporation. On the other hand, in the DNA-optimized transposase group, the expression rate of 1G4 TCR was 49.2% and 50.8%, while in the mRNA-optimized transposase group it was 79.0% and 79.0%, confirming that the expression rate of 1G4 TCR in the mRNA-optimized transposase group was about 29% or more higher. Furthermore, after 14 days of culture, the expression rate of 1G4 TCR in the DNA-optimized transposase group was 54.7% and 57.0%, while in the mRNA-optimized transposase group it was 77.2% and 73.5%, confirming that, similar to the results obtained on day 7, the expression rate in the mRNA-optimized transposase group was about 20% or more higher.

[0342] 4-2. Confirm the proportion of memory T cells in 1G4 TCR-T cells based on the optimized transposase gene delivery type.

[0343] Based on the optimized transposase gene delivery pattern, the proportion of memory T cells in 1G4 TCR-expressing T cells (CD3+1G4+ T cells) was analyzed using CD45RO and CD62L markers. When using CD45RO and CD62L markers to distinguish memory T cells, CD45RO-CD62L-T cells were classified as T cells. EFF (Effective T cells), CD45RO-CD62L+ T cells are classified as T cells. SCM (Stem cell-like memory T cells), CD45RO+CD62L-T cells are classified as T cells. EM (Effective memory T cells), CD45RO+CD62L+ T cells are classified as T cells. CM (Central memory T cells). The location of each memory T cell in the quadrant diagram is as follows: Figure 19 As shown.

[0344] To compare the proportion of memory T cells based on the delivery type of the transposase gene, the proportion of memory T cells was confirmed on days 7 and 14 after transformation with the 1G4 TCR gene using a transposon vector. The results are as follows: Figure 20 As shown in Table 11, it was confirmed that on day 7 post-transformation, with DNA type optimized transposase, T SCM and T CM The proportion was approximately 97.0%, and approximately 96.0% in the case of mRNA-type optimized transposases similar to the DNA type. Furthermore, on day 14, it was confirmed that in the case of DNA-type optimized transposases, T... SCM and T CM The proportion was approximately 75.0%, and approximately 69.0% under the condition of mRNA-optimized transposases, indicating that T SCM and TCM Memory T cells were present in high proportions in both delivery types.

[0345] [Table 11]

[0346] 4-3.1 Confirmation of the ratio of CD4 to CD8 in G4 TCR-expressing T cells

[0347] To confirm the ratio of CD4 to CD8 in 1G4 TCR-expressing T cells based on the transposase gene delivery type, the ratio was confirmed on days 7 and 14 post-transformation. The results are as follows: Figure 21 As shown in Table 12, it was confirmed that on day 7 post-transformation, the proportions of CD8+ and CD4+ were 86.3% and 20.8% with the DNA-optimized transposase, respectively, and 82.1% and 20.5% with the mRNA-optimized transposase, respectively. Furthermore, on day 14, the proportions of CD8+ and CD4+ were approximately 80.2% and 20.3% with the DNA-optimized transposase, and approximately 82.5% and 17.8% with the mRNA-optimized transposase, respectively. This indicates that the proportion of CD8+ cells was high in both delivery types, with no difference between them.

[0348] [Table 12]

[0349] 4-4. Results of cytotoxicity assay based on fluorescent enzyme

[0350] For the in vitro killing assay of 1G4 TCR-T cells cultured for 14 days, T2-Luc cells were co-cultured with a peptide pulse of 100 μg / mL at ratios of 30:1, 10:1, 3:1, and 1:1 (effective cells:target cells). The luciferase levels detected when target cells were killed were converted to percentage (%) values ​​to calculate the luciferase expression rate. Therefore, as... Figure 22As shown in Table 13, in the "no E / P (negative control) group" which did not express 1G4 TCR, the expression rate (%) determined by luciferase was less than 7.0% at 30:1 and undetectable at 10:1, 3:1, and 1:1. In the group using mRNA-optimized transposases to introduce 1G4 TCR using a transposon system, the expression rate (%) determined by luciferase was 67.3%, 48.1%, and 13.4% at 30:1, 10:1, and 3:1, respectively, but undetectable at 1:1. In the group using DNA-optimized transposases, the expression rate (%) determined by luciferase was 54.2% and 32.2% at 30:1 and 10:1, respectively, but undetectable at 3:1 and 1:1. Therefore, it was confirmed that the cytotoxic activity of 1G4 TCR-T cells delivered with mRNA-optimized transposases was greater than 10%.

[0351] [Table 13]

[0352] As described above, GFP gene expression was confirmed based on transposase delivery type (mRNA and DNA) when the gene was delivered to PBMC cells using a transposon system. Following Neon electroporation transformation, GFP gene expression was confirmed by FACS analysis on days 7 and 14 post-culture. The results confirmed that on day 7, the GFP expression rate of the mRNA-optimized transposase group was approximately 10% higher than that of the DNA-optimized transposase group, and on day 14, the expression rate of the mRNA-optimized transposase group was approximately 5% higher. Furthermore, after transformation with the 1G4 TCR gene using MaxCyte electroporation (a different type of electroporation device), 1G4 TCR gene expression was confirmed by FACS analysis on days 7 and 14 post-culture. The results confirmed that the 1G4 TCR gene was not expressed in the negative control group (EP only) which underwent electroporation only. On the other hand, it was confirmed that the expression of the 1G4 TCR gene in the mRNA-optimized transposase group was approximately 29% higher on day 7 post-transformation than that in the DNA-optimized transposase group. Furthermore, it was confirmed that after 14 days of culture, the expression rate of the mRNA-optimized transposase group was approximately 20% higher than that of the DNA-optimized transposase group. Additionally, it was confirmed that regardless of the gene delivery type, T... SCM and T CMThere was no difference in the proportion of memory cells and the proportions of CD4 and CD8. Furthermore, in the in vitro killing assay of 1G4 TCR-T cells cultured for 14 days, T2-Luc cells were co-cultured with 100 μg / mL peptide pulses at ratios of 30:1, 10:1, 3:1, and 1:1 (effective cells: target cells). The luciferase detected when target cells were killed was converted to a percentage (%) value for calculating the luciferase expression rate. The results confirmed that no 1G4 TCR expression was observed in the "no E / P (negative control) group," and no luciferase was detected under any conditions. The cytotoxicity test results of the mRNA-optimized transposase group were 10% or more higher than those of the DNA-optimized transposase group under all ratio conditions. Based on these results, it is confirmed that higher gene expression rates and cytotoxic effects are achieved when the transposase gene is delivered in the mRNA form of the transposon system rather than in DNA form.

[0353] Example E. Confirmation of pBat transposase gene delivery efficiency after adding nuclear localization sequence (NLS) gene

[0354] After being translated into proteins, transposases enter the cell nucleus. Therefore, the NLS gene was added to the 5' or 3' end of the transposase to improve its efficiency in entering the cell nucleus, and then the gene delivery efficiency was confirmed.

[0355] SV40 and C-myc were selected as NLS and added to the 5' or 3' end of the optimized transposase. The DNA sequences of SV40 and C-myc were also optimized for good expression in T cells.

[0356] [Table 14]

[0357] like Figure 22 and Figure 23 As shown, optimized SV40 or C-myc is added to the 5' or 3' end of the optimized transposase (SEQ ID NO:2) to optimize the transposase.

[0358] 1. Confirmation of GFP gene delivery efficiency

[0359] The efficiency of GFP gene delivery optimized by adding NLS to the 5' or 3' end was confirmed. To confirm GFP gene delivery efficiency, Jurkat T cell line (1x10⁻¹²) was used. 5 Neon transfection (electroporation) was performed in 24-well plates at 1,600 V, 10 ms, and 3 pulses. The transposons and DNA types shown in Table 15 below were optimized for transposase at a ratio of 1 μg:1 μg. GFP expression levels in cells were confirmed using fluorescence microscopy and FACS at 1, 7, and 14 days post-transfection. One day after Neon transfection, Jurkat cells cultured in 24-well plates were transferred to T25 flasks.

[0360] [Table 15]

[0361] like Figure 24a and Figure 24b As shown, one day after electroporation, there was no difference depending on the type and location of the NLS (5' or 3'). Figure 25a and Figure 25b As shown, it was confirmed that 7 days after electroporation, when C-myc was located at the 5' end, the overall GFP expression rate and high-intensity GFP expression rate were high. The GFP expression rate at 14 days after electroporation was... Figure 26 The same as shown.

[0362] To confirm the efficiency of the NLS transposase in Jurkat cells, FACS analysis was performed on GFP expression at 1 and 7 days after electroporation. The results are as follows: Figure 27 As shown, it was confirmed that 3'SV showed the highest GFP expression 1 day after electroporation, but 5'Myc showed the highest expression 7 days after electroporation, followed by 3'Myc and the optimized transposase without NLS. Furthermore, as... Figure 28 As shown, the high-intensity GFP expression rate was 8% in the optimized transposase without NLS, 17% in 5'Myc, and 11% in 3'Myc. The transposase with 5'Myc insertion showed the best stable transfection efficiency in Jurkat cells.

[0363] Furthermore, Jurkat cells expressing GFP on day 14 of electroporation were sorted on day 15 and cultured for an additional 9 days. The results were as follows: Figure 29a and Figure 29b As shown, it was confirmed that GFP-expressing cells stably maintained a GFP expression rate of 95%.

[0364] 2.1 Confirmation of G4 TCR gene delivery efficiency

[0365] The delivery efficiency of the 1G4TCR gene was confirmed by adding an NLS-optimized transposase to the 5' or 3' end of PBMCs. Specifically, PBMCs (LK048, 17011) stored in liquid nitrogen (LN2) were used, and OC100X2 (resting T cell 14-3 protocol) with 5x10 6 Transfection (electroporation) was performed using PBMC / 50 μL opti-MEM buffer and MaxCyte. The plasmid, transposon vector, and DNA type-optimized transposase shown in Table 16 were used at a transposon:transposase ratio of 8 μg:2 μg. FACS analysis was performed at 7 and 14 days post-transfection.

[0366] [Table 16]

[0367] The delivery efficiency of the 1G4 TCR gene in PBMCs was confirmed using 8 μg / well transposons and 2 μg / well transposase. Figures 30a to 30d As shown, FACS analysis performed seven days post-transfection confirmed a high 1G4 TCR expression rate in the C-myc group. Figures 31a to 31d As shown, the FACS analysis performed 14 days after transfection confirmed that, similar to day 7, the expression rate of 1G4 TCR in the C-myc group was very high.

[0368] Example F. Comparison of gene delivery efficiency between pBat transposon and lentivirus using CD19 CAR

[0369] The gene delivery efficiency of the non-viral vector pBat transposon and the viral vector lentivirus was compared.

[0370] [method]

[0371] 1. DNA and cells

[0372] To compare the non-viral vector pBat transposon with the viral vector lentivirus, pBat transposon plasmid vector and pBat optimized transposase plasmid vector (SEQ ID NO:14) were used as the non-viral vector pBat transposon, and lentivirus was produced using lentivirus CD19CAR transfer plasmid vector and lentivirus packaging vector.

[0373] PBMC (LK032 / LK053), Lenti-X 293T (Takara, catalog number 632180, batch number AIY0002S), Jurkat, clone E6-1 (ATCC, catalog number TIB-152, batch number 70017560), NALM6, and GFP-luciferase reporter cell lines were used as target cells.

[0374] 2. Preparation of pBat CD19-CAR plasmid vector

[0375] To compare gene delivery efficiency in CAR-T production with lentiviral transfer vectors, the following cloning method was used: the EGFP gene portion in the pBat transposon plasmid vector containing EGFP was replaced with CD19-CAR to produce the pBat CD19-CAR plasmid vector.

[0376] 1) Use primers containing the CD19-CAR gene as templates to perform PCR on the CD19-CAR gene to be used as an insert, and then load the PCR product onto an agarose gel to elute the DNA of the desired size band.

[0377] 2) Elute DNA of the desired size by treating with restriction enzymes BstB I and EcoRI from a pBat transposon plasmid vector containing EGFP.

[0378] 3) Use the DNA obtained in 1) and 2) to perform Gibson assembly cloning.

[0379] 4) In order to identify pBat CD19 CAR in the cloned clones, the restriction enzyme EcoR V was used to perform enzyme mapping, select candidate clones and finally sequence them.

[0380] 3. PBMC Activation

[0381] 1) RPMI medium (RPMI + 10% FBS + 1X P / S) was prepared by preheating in a 37°C water bath.

[0382] 2) Remove PBMC LK032 and LK053 from the nitrogen tank and thaw them quickly in a 37°C water bath.

[0383] 3) Prepare two 50mL tubes, add 30mL of culture medium to each tube, and put the thawed cells from the PBMC LK032 and PBMCLK053 vials into each 50mL tube.

[0384] 4) Centrifuge at 1,500 rpm for 5 minutes, remove the supernatant, suspend the cells in 40 mL of RMPI medium, and count the cells.

[0385] 5) 1.2 x 10 8 Add 1 PBMC to a T175 flask, add RPMI medium to prepare 1.2 x 10⁻⁶ ppm. 8 / 120mL.

[0386] 6) Add IL-2 to a final concentration of 20 IU / mL, every 2.0 x 10 7 Add 100 μL TransAct TM (Improve IL-2 from 1.0 x 10⁻⁶ using culture medium) 6 IU / mL diluted to 1.0 x 10⁻⁶ 5 (IU / mL).

[0387] 7) Incubate for two days in a 37°C, 5% CO2 incubator.

[0388] 4. PBMC electroporation

[0389] As described below, a nonviral transposon system containing the CD19 CAR gene is delivered into activated PBMCs.

[0390] 1) Collect the activated PBMCs in a conical tube, centrifuge at 300x g for 10 minutes, and remove the supernatant.

[0391] 2) Suspend the cell clumps in 20 mL of culture medium (ALYS505N-O+3% HS) and count the cells.

[0392] 3) After centrifuging at 1,500 rpm for 5 minutes at room temperature, remove the supernatant and suspend the cell clumps in 20 mL L PBS.

[0393] 4) After centrifuging at 1,500 rpm for 5 minutes at room temperature, the supernatant was completely removed.

[0394] 5) Remove the supernatant and suspend the cell clumps in 5 mL of Opti-MEM medium.

[0395] 6) After centrifuging at 1,500 rpm for 5 minutes at room temperature, the supernatant is completely removed.

[0396] 7) Resuspend the cell clumps in 50 μL of warm Opti-MEM medium to a final volume of 5.0 x 10⁻⁶. 6 / 50μL.

[0397] 8) As shown in Table 17 below, the transposon vector and transposase vector were added to the tubes in 7) at a rate of 5 μg per well. However, no vector was added to the control group.

[0398] [Table 17] **Each group n=2 experiments were conducted. For LK053, the experiments were conducted only under the condition of 200 IU / mL IL-2.

[0399] 9) Take 5 x 10 from 7) 6 Carefully add 1 cell / 50μL cell suspension to the OC100X2 unit to avoid generating bubbles.

[0400] 10) For electroporation, select the resting T-cell 14-3 protocol in MaxCyte STx.

[0401] 11) Insert the OC100X2 assembly from 9) into the chamber in the STx and perform electroporation by performing this procedure.

[0402] 12) After electroporation, transfer the cell suspension from the OC100x2 assembly to a T25 flask (5 x 10). 6 (1 cell / 50μL / well).

[0403] 13) Wash the OC100X2 wells with 50 μL of Opti-MEM medium and add it to each well of the plate in step 11).

[0404] 14) Provide a 20-minute recovery time in a 37°C, 5% CO2 incubator.

[0405] 15) Carefully add 10 mL of complete culture medium (ALYS505N-O + 3% HS + 200 IU / mL IL-2) to a T25 flask containing PBMC, then place the resulting mixture back into a 37°C, 5% CO2 incubator and incubate for two days.

[0406] 16) Two days after electroporation, 20 mL of culture medium (ALYS505N-O+3%HS+1x P / S) was added to the control group, and 10 mL of culture medium was added to the CAR group. Then, IL-2 was added to a final concentration of 200 IU / mL or 400 IU / mL and cultured in a 37℃, 5% CO2 incubator.

[0407] 17) Five days after electroporation, add IL-2 to a final concentration of 200 IU / mL or 400 IU / mL and incubate at 37°C in a 5% CO2 incubator.

[0408] 18) Seven days after electroporation, the cultured cells were resuspended, and 500 μL of the cell suspension was transferred to a new tube for FACS analysis. The remaining cell suspension was added to fresh culture medium (ALYS505N-O + 3% HS + 1x P / S) to a concentration of 5.0 x 10⁻⁶. 6 Add IL-2 to a final concentration of 200 IU / mL or 400 IU / mL, and incubate at 37°C in a 5% CO2 incubator.

[0409] 19) Nine days after electroporation, the cultured cells were resuspended, and half of the suspension was stored as stock solution. The supernatant was removed, and CS10 medium was added. The resulting mixture was stored in a cryogenic chamber for 1 day, and then transferred to a nitrogen tank.

[0410] 20) Add the remaining half of the suspension to fresh culture medium (ALYS505N-O + 3% HS + 1x P / S) until the concentration is 5.0x 10⁻⁶. 6 Add IL-2 to a final concentration of 200 IU / mL or 400 IU / mL and incubate at 37°C in a 5% CO2 incubator.

[0411] 21) Twelve days after electroporation, cells in suspension culture were injected at a concentration of 5.0 x 10⁻⁶. 6Add fresh medium (ALYS505N-O + 3% HS + 1x P / S) to cells / mL. Add IL-2 to a final concentration of 200 IU / mL or 400 IU / mL and incubate at 37°C in a 5% CO2 incubator.

[0412] 22) Fourteen days after electroporation, the cultured cells were resuspended, and 500 μL of the cell suspension was transferred to new tubes for FACS analysis. 2.0 x 10⁻⁶ cells were harvested from each group. 7 Cells were subjected to in vitro killing assays and cultured in 20 mL of resting medium (RPMI 1640 + 10% FBS + 1x P / S). To prepare the stock solution from the remaining suspension, the supernatant was removed and CS10 medium was added. The resulting mixture was stored in a cryogenic chamber for 1 day and then transferred to a nitrogen tank.

[0413] 5. Lentiviral transduction

[0414] As described below, a lentivirus containing the CD19-CAR gene is delivered into activated PBMCs.

[0415] 1) Collect the activated PBMCs in a 50 mL conical tube and centrifuge at 300 x g for 10 minutes at room temperature.

[0416] 2) Remove the supernatant, suspend the cell clumps in 20 mL of culture medium (RPMI 1640 + 10% FBS + 1x P / S), and count the cells.

[0417] 3) Resuspend PBMCs in the culture medium to 5.0 x 10⁻⁶. 6 / 2mL.

[0418] 4) Hole the required number of holes at a size of 5.0 x 10 6 2 mL per well was inoculated into a 12-well plate.

[0419] 5) Each well was treated with 10 μg / mL protamine and lentivirus, with a multiplicity of infection (MOI) of 4.

[0420] 6) Perform rotary cutting at 1,200 x g for 90 minutes.

[0421] 7) After centrifugation, transfer the PBMCs from each well to a T75 flask.

[0422] 8) Add the culture medium to the T75 flask to make the total volume 10 mL. The experimental groups are shown in Table 18 below.

[0423] [Table 18] No. Group Cell count activation Lentiviral IL-2 1 Activation only <![CDATA[5.0x 10 6 Individual cells]]> <![CDATA[TransAct TM ]]> - 200 IU / mL 2 Activation only <![CDATA[5.0x 10 6 Individual cells]]> <![CDATA[TransAct TM ]]> - 400 IU / mL 3 Lentiviral CD19 CAR <![CDATA[5.0x 10 6 Individual cells]]> <![CDATA[TransAct TM ]]> 4MOI 200 IU / mL 4 Lentiviral CD19 CAR <![CDATA[5.0x 10 6 Individual cells]]> <![CDATA[TransAct TM ]]> 4MOI 400 IU / mL

[0424] 9) Add IL-2 to a final concentration of 200 IU / mL or 400 IU / mL and incubate at 37°C in a 5% CO2 incubator.

[0425] 10) Two days after transduction, suspend the cultured cells and add fresh culture medium (RPMI 1640 + 10% FBS + 1x P / S) to the cell suspension until the concentration is 5.0x 10⁻⁶. 6 / mL. Add IL-2 to a final concentration of 200 IU / mL or 400 IU / mL and incubate at 37°C in a 5% CO2 incubator.

[0426] 11) Five days after transduction, add the same amount of fresh culture medium (RPMI 1640 + 10% FBS + 1x P / S), add IL-2 to a final concentration of 200 IU / mL or 400 IU / mL, and incubate at 37°C in a 5% CO2 incubator.

[0427] 12) Seven days after transduction, suspend the cultured cells and transfer 500 μL of the cell suspension to a new tube for FACS analysis. Add fresh medium (RPMI 1640 + 10% FBS + 1x P / S) to the remaining cell suspension to a concentration of 5.0 x 10⁻⁶. 6 Add IL-2 to a final concentration of 200 IU / mL or 400 IU / mL and incubate at 37°C in a 5% CO2 incubator.

[0428] 13) Nine days after transduction, the cultured cells were resuspended, and half of the suspension was prepared as the stock solution. The supernatant was removed, and CS10 medium was added. The resulting mixture was stored in a cryogenic freezer for 1 day, and then transferred to a nitrogen tank. Fresh medium (RPMI 1640 + 10% FBS + 1x P / S) was added to the remaining half of the suspension to a concentration of 5.0 x 10⁻⁶. 6 Add IL-2 to a final concentration of 200 IU / mL or 400 IU / mL and incubate at 37°C in a 5% CO2 incubator.

[0429] 14) Twelve days after transduction, suspend the cultured cells and add fresh medium (RPMI 1640 + 10% FBS + 1x P / S) to a concentration of 5.0 x 10⁻⁶. 6 Add IL-2 to a final concentration of 200 IU / mL or 400 IU / mL and incubate at 37°C in a 5% CO2 incubator.

[0430] 15) Fourteen days after transduction, the cultured cells were resuspended, and 500 μL of the cell suspension was transferred to a new tube for FACS analysis. 2.0 x 10⁻⁶ cells were harvested from each group.7 Cells were subjected to in vitro killing assays and cultured in 20 mL of resting medium (RPMI 1640 + 10% FBS + 1x P / S). To prepare the stock solution from the remaining suspension, the supernatant was removed and CS10 medium was added. The resulting mixture was stored in a cryogenic chamber for 1 day and then transferred to a nitrogen tank.

[0431] 6. FACS Analysis

[0432] FACS analysis was performed at 7, 10 and 14 days after electroporation.

[0433] 1) Suspend the cells in each group of flasks and transfer them to each 3 mL FACS tube.

[0434] 2) Centrifuge at 4°C and 1,500 rpm for 5 minutes, remove the supernatant, and resuspend the cells in 1 mL of washing buffer (PBS + 2% FBS).

[0435] 3) Repeat step 2) to wash again.

[0436] 4) Centrifuge at 4°C and 1,500 rpm for 5 minutes to remove the supernatant.

[0437] 5) Add 5 μL of human TruStain FcX + 95 μL of FACS buffer to each tube and incubate the resulting mixture at room temperature for 5 minutes.

[0438] 6) Each antibody (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, anti-CCR7, anti-CD62L, anti-FLAG TAG) was added in 2 μL per tube, and the resulting mixture was incubated in the dark at room temperature for 30 minutes.

[0439] 7) Add 1 mL of FACS buffer (PBS + 2% FBS) and centrifuge at 4°C and 1,500 rpm for 5 minutes.

[0440] 8) Remove the supernatant and resuspend the cells in 1 mL of FACS buffer (PBS + 2% FBS).

[0441] 9) Centrifuge at 4°C and 1,500 rpm for 5 minutes to remove the supernatant.

[0442] 10) Resuspend cells in 200 μL of FACS buffer (PBS + 2% FBS) containing 1x DAPI and perform FACS analysis.

[0443] 7. Luciferase-based cytotoxicity assay of CD19 CAR-T cells and GFP-luciferase reporter-NALM6 cells after co-culture

[0444] 1) Collect the GFP-luciferase reporter-NALM6 cells cultured in the T75 flask into a 50 mL tube.

[0445] 2) Centrifuge at room temperature and 1,500 rpm for 5 minutes.

[0446] 3) Remove the supernatant, suspend the cell clumps in 5 mL of RPMI medium, and count the cells.

[0447] 4) Add RPMI medium (10% FBS + 1% P / S) to a final volume of 2.0 x 10⁻⁶. 4 Prepare GFP-luciferase reporter-NALM6 cells per 100 μL.

[0448] 5) After counting the two types of resting CD19 CAR-T cells (cells prepared in 6. and cells prepared in 7.), the prepared cells had a concentration of 6.0 x 10⁻⁶. 5 / 100μL.

[0449] 6) such as Figure 33 As shown, 100 μL of RPMI medium (10% FBS + 1% P / S) was placed in channels B to D and F to H of two white 96-well plates beforehand, and 150 μL of 9.0 x 10⁵ P / S medium was added to each plate. 5 CD19 CAR-T cells were placed in channels A and E. Then, PBMCs were serially diluted 3-fold using multichannel pipettes to achieve NALM6 / PMC ratios of 30:1, 10:1, 3:1, and 1:1 (plate 1: transposon system; plate 2: lentiviral system).

[0450] 7) such as Figure 33 As shown, NALM6 cells prepared in step 4) were added to channels A through D at a rate of 100 μL each, and co-cultured at a rate of 200 μL / well.

[0451] 8) Add 100 μL of complete RPMI to the “PBMC Only” group (channels E to H) to adjust to 200 μL / well.

[0452] 9) such as Figure 33 As shown, 100 μL of “NALM6 only” cells and 100 μL of complete RPMI were added to channels 11 and 12 to adjust to 200 μL / well.

[0453] 10) To facilitate lysis, add 20 μL of 10% Triton-X to channel 12 and mix thoroughly.

[0454] 11) Incubate at 37°C in a 5% CO2 incubator for 4 hours to carry out the reaction.

[0455] 12) Dissolve the Bright-Glo reagent mixture (100 mL Bright Glo luciferase assay buffer + substrate) in the required amount in a 37°C, 5% CO2 incubator.

[0456] 13) After fully suspending the mixture in each well of the 96-well plate for co-culture, take out 100 μL.

[0457] 14) Add 100 μL of Bright-Glo reagent to each well, cover each well with aluminum foil, and let the mixture stand at room temperature for two minutes.

[0458] 15) Install the 96-well plate on the spectrophotometer and measure the luminescence.

[0459] [result]

[0460] 1. Results of pBat CD19 CAR plasmid cloning

[0461] To clone the pBat CD19 CAR plasmid vector, BstBI and EcoRI were processed, and then the CD19 CAR gene to be used as the insert was assembled using Gibson. Furthermore, to confirm the success of the cloning, [further details are needed]. Figure 34 As shown, enzyme mapping was performed using the restriction enzyme EcoR V, then candidate clones were selected and finally identified by sequencing.

[0462] 2. Confirmation of total cell count in culture

[0463] To generate CD19 CAR-T cells, genes were delivered to PBMCs via transduction and electroporation using lentivirus and transposon methods, respectively, and the cells were cultured for 7 and 14 days. Subsequently, the total number of cultured T cells and cell viability were confirmed. Regarding the total cell count, as shown... Figure 35 As shown in Table 19, it was confirmed that compared with the control group not treated with the vector in this experiment, the survival rate induced by electroporation in the transposon vector-treated group tended to decrease on day 7, but the cell proliferation rate increased over time.

[0464] [Table 19]

[0465] like Figure 36 As shown in Table 20, on day 7, the cell survival rate of lentivirus was 85.8% to 94.4%, and that of transposon was 87.3% to 94.4%; on day 14, the cell survival rate of lentivirus was 77.7% to 96.2%, and that of transposon was 91.4% to 93.4%, confirming that the survival rate of T cells using lentiviral vectors was slightly lower than that of T cells using transposon vectors.

[0466] [Table 20]

[0467] 4. Comparison of CD19 CAR protein expression based on gene delivery vectors

[0468] CD19-CAR expression was confirmed using an anti-FLAG tag antibody via FACS, where the FLAG tag protein is expressed by the FLAG tag gene located between the leader sequence and CD19scFv in the CD19-CAR gene. Figure 37 As shown, the analysis method was performed by gating in the order of monomers, live cells, lymphocytes, CD3+ T cells and FLAG+ T cells to confirm the delivery and expression efficiency of the CD19-CAR gene to CD3+ T cells.

[0469] 4-1.LK032 PBMC

[0470] After transforming LK032 PBMCs with the CD19-CAR gene using lentivirus and transposon vectors, the proportion of CD3+ T cells expressing FLAG (CD19-CAR) was confirmed on day 7 of culture. Results, as follows... Figure 38 As shown in Table 21, FLAG (CD19-CAR) was confirmed not to be expressed in the negative control group "Activation Only" (where activation was performed only with each vector) and the negative control group "EP Only" (where electroporation was performed only). In the groups transformed with each vector and cultured for 7 days with 200 IU / mL IL-2, the proportions of cells expressing FLAG (CD19-CAR) using lentivirus and transposon vectors were 41.0% and 54.6%, respectively, indicating a approximately 14% higher proportion of cells using transposon vectors. In the groups cultured with 400 IU / mL IL-2, these proportions were 43.4% and 59.6%, respectively, indicating a approximately 15% higher proportion of cells using transposon vectors. Furthermore, after 14 days of culture, the proportions of cells expressing FLAG (CD19-CAR) in the group cultured with 200 IU / mL IL-2 were 67.30% and 61.0%, indicating a slightly lower proportion of transposon vectors. However, in the group cultured with 400 IU / mL IL-2, the proportions were 62.5% and 63.5%, almost similar to each other. On the other hand, it has been confirmed that in cells expressing FLAG, the proportion of cells expressing FLAG with high intensity using transposon vectors was high on day 7, while this proportion was high for lentiviral vectors on day 14.

[0471] [Table 21]

[0472] 4-2.LK053 PBMC After transforming LK053 PBMCs with the CD19 CAR gene using lentivirus and transposon vectors, the proportion of CD3+ T cells expressing FLAG (CD19 CAR) was confirmed on day 7 of culture. Results, as follows... Figure 39 As shown in Table 22, FLAG (CD19-CAR) was confirmed not to be expressed in the negative control group "Activation Only" (where activation was performed only with each vector) and the negative control group "EP Only" (where electroporation was performed only). In the groups cultured for 7 days after transformation with 200 IU / mL IL-2, the proportions of cells expressing FLAG (CD19-CAR) using lentivirus and transposon vectors were 44.3% and 59.0%, respectively, indicating a proportion approximately 15% higher using transposon vectors. Furthermore, after 14 days of culture, the proportions of cells expressing FLAG (CD19-CAR) in the groups cultured with 200 IU / mL IL-2 were 49.1% and 57.3%, respectively, indicating a proportion approximately 8% higher using transposon vectors. Moreover, unlike with LK032, a high proportion of cells expressing FLAG using transposon vectors was confirmed at days 7 and 14.

[0473] [Table 22]

[0474] 5. Confirm the proportion of memory T cells based on the gene delivery vector.

[0475] The proportion of memory T cells in T cells (CD3+ cells) transformed with two different vectors was analyzed using CD45RA and CD62L markers. When memory T cells were distinguished using CD45RA and CD62L markers, CD45RA+CD62L-T cells were classified as T cells. EFF (Effective T cells), CD45RA-CD62L+ T cells are classified as T cells. EM (Effective memory T cells), CD45RA-CD62L+ T cells are classified as T cells. CM (Central memory T cells), CD45RA+CD62L+ T cells are classified as T cells. SCM (Stem cell-like memory T cells). The location of each memory T cell in the quadrant diagram is as follows: Figure 40 As shown.

[0476] 5-1.LK032 PBMC

[0477] After transforming LK032PBMCs with the CD19 CAR gene using lentiviral and transposon vectors, the proportion of T-cell memory phenotypes in total cultured cells was confirmed on days 7 and 14. Results, as follows... Figure 41As shown in Table 23, in the negative control group "activation only" (where activation was performed only with each carrier) and the negative control group "EP only" (where electroporation was performed only), T SCM and T CM The sum of these proportions was similarly confirmed to be on average 56% to 65%. In the case of lentiviral vectors, in all groups cultured on day 7 post-transformation with 200 IU / mL and 400 IU / mL IL-2, T SCM and T CM The sum of the proportions was confirmed to be approximately 61%, while in the case of transposon vectors, the T groups cultured with 200 IU / mL and 400 IU / mL IL-2 showed a similar ratio. SCM and T CM The combined proportions were confirmed to be approximately 80% to 82%, about 20% higher than that in the group using lentiviral vectors. Furthermore, on day 14, in the case of lentiviral vectors, T... SCM and T CM The sum of these proportions was confirmed to be approximately 43% to 48%, while in the case of transposon carriers, T SCM and T CM The sum of the proportions was confirmed to be slightly high, ranging from 47% to 56%, indicating that in the group using transposon vectors, T SCM and T CM The overall proportion of memory T cells is very high.

[0478] [Table 23]

[0479] 5-2.LK053 PBMC

[0480] After transforming LK053PBMCs with the CD19 CAR gene using lentiviral and transposon vectors, the proportion of T-cell memory phenotypes in total cultured cells was confirmed on days 7 and 14. Results, as follows... Figure 42 As shown in Table 24, on day 7, in the negative control group of the lentiviral vector, T SCM and T CM The combined proportions were approximately 46% to 57%, and approximately 65% ​​in the negative control group of the transposon vector, confirming the presence of T in the transposon vector. SCM and T CM The proportion of memory T cells was high. Furthermore, T cells were present in the negative control group on day 14. SCM and T CM The sum of these proportions is approximately 22% to 33% in the case of lentiviral vectors and approximately 43% in the case of transposon vectors, confirming the presence of T in transposon vectors. SCM and T CMThe proportion of memory T cells was high. Furthermore, in the case of lentiviral vectors in the experimental groups, on day 7, in all groups cultured with 200 IU / mL and 400 IU / mL IL-2, T cells... SCM and T CM The proportion of T cells in the transposon vector group was confirmed to be approximately 57% to 62%, while in the case of the transposon vector, this proportion was confirmed to be approximately 75% in the group cultured with 200 IU / mL IL-2, about 15% higher than the group using the lentiviral vector. Furthermore, at day 14, this proportion was confirmed to be approximately 35% to 42% in the case of the lentiviral vector and 46% in the case of the transposon vector, slightly higher than the proportion in the group using the lentiviral vector, indicating that the T cells in the transposon vector group had a higher proportion of T cells. SCM and T CM The overall proportion of memory T cells is very high.

[0481] [Table 24]

[0482] 6. Identify memory T cells in T cells expressing FLAG (CD19-CAR).

[0483] Memory T cells transformed and expressing FLAG (CD19-CAR) using two delivery systems were identified. The results were as follows: Figure 43 As shown in Table 25, in the case of lentiviral vectors, both LK032 PBMC and LK053 PBMC showed T on day 7. SCM and T CM The sum of the proportions is approximately 59% to 65%, while in the case of transposon vectors, the sum of the proportions is approximately 78% to 85%, which is about 20% higher than the sum of the proportions in lentiviral vectors. However, on day 14, in the case of lentiviral vectors, the T in both LK053 and LK032... SCM and T CM The sum of the proportions is approximately 40% to 54%, while in the case of transposable carriers, the sum of the proportions is approximately 48% to 57%, confirming that the proportions of the two carriers are similar.

[0484] [Table 25]

[0485] 7. Confirm the ratio of CD4 to CD8 in T cells expressing FLAG (CD19-CAR).

[0486] To confirm the ratio of CD4 to CD8 in FLAG (CD19-CAR)-expressing T cells transformed using two delivery systems, this ratio was confirmed on days 7 and 14 post-transformation. The results are as follows: Figure 44 and Figure 45As shown in Table 26, it was confirmed that in the case of lentiviral vectors, the proportions of CD8+ and CD4+ on day 7 post-transformation were 28%–38% and 45%–54%, respectively, in all groups cultured with 200 IU / mL and 400 IU / mL IL-2. In the case of transposon vectors, the proportions were 56%–59% and 53%–59%, respectively, confirming that the proportion of CD8+ was 5%–10% higher than in the lentiviral case. Furthermore, it was confirmed that in the case of lentiviral vectors, the proportions of CD8+ and CD4+ on day 14 were approximately 39%–51% and 58%–75%, respectively, while in the case of transposon vectors, the proportions were approximately 64%–72% and 57%–61%, respectively, indicating that the proportion of CD8+ was approximately 20%–25% higher than in the lentiviral case.

[0487] [Table 26]

[0488] 8. Results of in vitro lethality assay

[0489] For the in vitro killing assay of CAR-T cells cultured for 14 days, co-culture with GFP-luciferase reporter-NALM6 cells was performed at ratios of 30:1, 10:1, 3:1, and 1:1 (effective cells:target cells). The luciferase levels detected when target cells were killed were converted to percentage (%) values ​​to calculate the luciferase expression rate. Results are as follows: Figure 46 As shown in Table 27, in the case of lentiviral vectors, at ratios of 30:1, 10:1, 3:1, and 1:1, the luciferase expression rates (%) in the "non-transduction (negative control) group" without CD19-CAR expression were approximately 5.3% to 17.2%, 3.4% to 8.9%, 1.0% to 7.4%, and 0% to 9.3%, respectively. In contrast, at ratios of 30:1, 10:1, 3:1, and 1:1, the luciferase expression rates (%) in the lentivirally transduced groups were 52.8% to 55.9%, 41.5% to 44.6%, 21.4% to 23.6%, and 0% to 5.2%, respectively, confirming a dose-dependent increase in CD19 CAR-T cells.

[0490] [Table 27]

[0491] Furthermore, in the case of transposable carriers, such as Figure 47As shown in Table 28, under ratios of 30:1, 10:1, 3:1, and 1:1, the luciferase expression rates (%) in the "no E / P (negative control) group" without CD19 CAR expression were 7.8% to 40.8%, 9.9% to 29.9%, 0% to 17.3%, and 0% to 24.3%, respectively. In the transposon transfected group, under ratios of 30:1, 10:1, 3:1, and 1:1, the luciferase expression rates (%) were 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 summary, it was confirmed that, under all conditions, CD19 CAR-T cells generated using transposon vectors exhibited approximately 20% higher in vitro killing activity against actual target cells than CD19 CAR-T cells generated using lentiviruses.

[0492] [Table 28]

[0493] As discussed above, the performance of the pBat transposon vector was confirmed by comparing gene delivery efficiency and the proportion of CD19 CAR-expressing cells during culture between the third-generation vector lentivirus (a widely used viral vector whose stability has been confirmed by the application of a self-inactivation (SIN) vector system) and the non-viral vector pBat transposon vector. The results showed that when PBMCs transformed with the CD19 CAR gene using both lentiviral and transposon vectors were cultured for 14 days, the cell viability of T cells generated using the lentiviral vector was slightly lower than that generated using the transposon vector. Furthermore, in both LK032 and LK053 PBMCs, on day 7 of culture, the proportion of cells expressing FLAG (CD19-CAR) in CD3+ T cells was approximately 15% higher in the transposon vector group, and on day 14, the FLAG (CD19-CAR) expression rate in the transposon vector group was similar to or slightly higher than that in the lentiviral vector group. Furthermore, the proportion of memory T cells in T cells (CD3+ and FLAG+ T cells) transformed with the two vectors was analyzed using CD45RA and CD62L markers. The results confirmed that both LK032 PBMCs and LK053 PBMCs using transposon vectors had a higher proportion of memory T cells. SCM and T CMThe proportion of memory T cells was higher. As a result of confirming the proportion of CD4 and CD8 T cells in FLAG (CD19 CAR)-expressing T cells, the proportion of CD8+ T cells in the transposon vector was approximately 5% to 10% higher than that in lentivirus on day 7 and approximately 20% to 25% higher on day 14. Finally, as a result of confirming the in vitro killing activity of CAR-T cells cultured for 14 days, it was confirmed that under all conditions, T cells produced with the transposon vector exhibited approximately 20% higher in vitro killing activity against actual target cells than T-cells produced with lentiviral vectors. Based on the above results, the pBat transposon vector was determined to be a viable gene delivery vector that can replace lentiviral vectors.

[0494] The above description of the present invention is for illustrative purposes only. Those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical concept or essential characteristics of the invention. Therefore, it should be understood that the above embodiments are exemplary in all respects and not restrictive.

[0495] In Table 29 below, the bold portion of SEQ ID NO:14 is the sequence of SEQ ID NO:2; the bold portion of SEQ ID NO:15 is the sequence of SEQ ID NO:3; the underlined portion of SEQ ID NO:16 is the sequence of SEQ ID NO:9, and the bold portion is the sequence of SEQ ID NO:2; the underlined portion of SEQ ID NO:17 is the sequence of SEQ ID NO:9, and the bold portion is the sequence of SEQ ID NO:2; the underlined portion of SEQ ID NO:18 is the sequence of SEQ ID NO:11, and the bold portion is the sequence of SEQ ID NO:2; the underlined portion of SEQ ID NO:19 is the sequence of SEQ ID NO:11, and the bold portion is the sequence of SEQ ID NO:2; the underlined portion of SEQ ID NO:20 is the sequence of SEQ ID NO:9, and the bold portion is the sequence of SEQ ID NO:2; the underlined portion of SEQ ID NO:21 is the sequence of SEQ ID NO:9, and the bold portion is the sequence of SEQ ID NO:2; SEQ ID The underlined portion of NO:22 is the sequence of SEQ ID NO:11, and the bold portion is the sequence of SEQ ID NO:2; the underlined portion of SEQ ID NO:23 is the sequence of SEQ ID NO:11, and the bold portion is the sequence of SEQ ID NO:2.

[0496] [Table 29]

[0497] [Industrial Applicability]

[0498] This invention relates to a highly active transposase protein of a transposon system and its uses, and can be effectively applied to the development of genome-modified cell lines expressing various genes because it can achieve efficient gene delivery by enhancing the activity of transposases.

[0499] Furthermore, when T cells are genetically modified using the hyperactive transposase according to the present invention, cytotoxic T (CD8) cells exhibit antitumor activity. + The proportion of T cells and memory T cells with enhanced in vivo persistence, such as T3 cells. CM (Central memory T cells) and T SCM With the increased proportion of stem cell-like memory T cells, the transposon system of the present invention is expected to be able to generate TCR-T cells and CAR-T cells with excellent in vivo persistence, indicating industrial applicability.

Claims

1. A transposase expression vector comprising a nucleic acid sequence encoding a transposase represented by SEQ ID NO:2 or SEQ ID NO:

3.

2. The transposase expression vector according to claim 1, wherein the nucleic acid sequence encoding the transposase further includes a nucleic acid sequence encoding a nuclear localization signal.

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 represented by SEQ ID NO:4 to 11.

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

5. mRNA encoding a transposase, said transposase being shown in SEQ ID NO:

12.

6. The mRNA of claim 5, wherein the mRNA is generated by in vitro transcription using the transposase expression vector of 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 a transposase represented by SEQ ID NO:

3.

8. A transposase expressed by the transposase expression vector as described in claim 1 or the mRNA encoding the transposase as described in claim 5.

9. The transposase of claim 8, wherein when the transposase expression vector is a transposase expression vector containing a nucleic acid sequence encoding the transposase represented by SEQ ID NO:3, the transposase is expressed by mRNA generated by in vitro transcription using the transposase expression vector.

10. The transposase according to claim 8, wherein the transposase comprises the amino acid sequence represented by SEQ ID NO:

13.

11. A transposon system for delivering target DNA, the system comprising: a) A transposon vector containing inserted target DNA; as well as b) A transposase expression vector containing a nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 or SEQ ID NO:3, or mRNA encoding the transposase represented by SEQ ID NO:12, or The transposase as described in claim 8.

12. The transposon system of 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 delivering target DNA, the kit comprising: The transposon system for delivering target DNA as described in claim 11; And the instruction manual.

14. A cell wherein the following are introduced: a) A transposon vector containing inserted target DNA; and b) A transposase expression vector containing a nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 or SEQ ID NO:3, or mRNA encoding the transposase represented by SEQ ID NO:12, or The transposase as described in claim 8.

15. The cell of claim 14, wherein the target DNA is cleaved from the transposon vector by the transposase within the cell, and the cleaved 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 T cells, NK cells, B cells, dendritic cells, macrophages and mast cells.

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

18. The cell of claim 17, wherein the feeder cell is a cell that has been irradiated.

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

20. A method for inserting a target DNA sequence into the genome of a cell, the method comprising: The steps for introducing a) and b) into the cell: a) A transposon vector containing inserted target DNA; as well as b) A transposase expression vector containing a nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 or SEQ ID NO:3, or mRNA encoding the transposase represented by SEQ ID NO:12, or The transposase as described in claim 8.

21. The method of claim 20, wherein the introduction is performed via electroporation.

22. The method of claim 20, further comprising: Following the introduction step, the cells in which the transposon vector is inserted are co-cultured with feeder cells.

23. The method of claim 22, wherein the step of co-culturing the cells with feeder cells is performed immediately after the introduction step.

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