Nucleic acid for retrovirus vector production

Abstract

The present invention provides a nucleic acid construct that is for producing a retrovirus vector and includes each of the following sequences in this order from the 5' end: (a) a 5'LTR (long terminal repeat) sequence derived from a retrovirus including an exogeneous promoter sequence; (b) a packaging signal sequence (ψ) derived from a retrovirus; (c) a sequence derived from a nucleic acid that encodes a gag protein with a length of 183-227 bp; (d) a desired sequence or a multicloning site; and (e) a 3'LTR sequence derived from a retrovirus.

Description

Nucleic acids for retroviral vector production

[0001] The present invention relates to a nucleic acid construct for producing a retroviral vector used to express a desired gene in mammalian cells, a retroviral vector containing a transcript from the nucleic acid construct, a method for producing a gene-transfected cell using the vector, and a cell containing the nucleic acid construct.

[0002] Known methods for introducing genes into eukaryotes include the use of viral vectors, and techniques for introducing naked DNA by endocytosis, electroporation, or gene guns. Viral vectors are a technology that has been widely used in the field of gene therapy, from basic to clinical research. For example, adenoviral vectors are suitable for transiently expressing large amounts of a target gene in target cells, and retroviral vectors enable long-term stable expression due to their ability to stably integrate into host chromosomes. These vectors are expected to be used in the field of gene therapy for genetic diseases, and are also expected to be used in the field of transgenic animal production.

[0003] Retroviruses contain the gag / pol genes, which encode precursor proteins such as viral particle structural proteins, protease, reverse transcriptase, and integrase, and the env gene, which encodes the envelope glycoprotein. Both ends of these genes are flanked by a 5' long terminal repeat (LTR) and a 3' LTR, which are involved in the transcription of the viral genome, reverse transcription from the viral genome, and integration of the double-stranded DNA synthesized through reverse transcription into the host DNA. Gene transfer systems using retroviral particles are divided into one or two packaging constructs expressing the gag / pol and env genes and a transfer vector encoding RNA to be incorporated into retroviral vector particles, in order to disable self-replication while maintaining the infectivity of the viral particles. The transfer vector contains the LTR and a viral particle packaging signal sequence, and has most of the gag / pol genes and the env gene deleted, with, for example, a desired sequence inserted. The promoter sequence in the 5'LTR of the transfer vector motivates transcription of the RNA genome, and the transcript is packaged into viral particles by the packaging signal sequence.

[0004] To further reduce the possibility of retrovirus self-replication, self-inactivating (SIN) retroviral vectors have been developed in which the promoter sequence of the 3' LTR, which functions as a promoter on the chromosome of the target cell, has been deleted. In SIN vectors, transcriptional regulation of the desired gene is carried out by an internal promoter located on the 3' end side of the packaging signal sequence. Since sequences 5' end of the internal promoter are not transcribed in SIN vectors, the possibility of generating self-replicating viral particles is extremely low, making them safe. To date, vectors with enhanced safety have been reported (Non-Patent Document 1, Non-Patent Document 2).

[0005] There is a need for the development of safer and more efficient vectors that can be used to express desired genes in mammalian cells.

[0006] Mol. Ther., Vol. 25, No. 8, pp. 1790-1804 (2017) J. Virology, Vol. 73, No. 7, pp. 6171-6176 (1999)

[0007] An object of the present invention is to provide a retroviral vector for introducing an exogenous gene into a cell and expressing the gene more safely and efficiently.

[0008] As a result of extensive efforts to solve the above problems, the present inventors discovered a retroviral vector that can achieve both expression of an introduced gene and safety, and thus completed the present invention.

[0009] That is, to summarize the present invention, [1] a nucleic acid construct for producing a retroviral vector, comprising, in order from the 5' end: (a) a retroviral-derived 5' LTR (Long Terminal Repeat) sequence containing an exogenous promoter sequence, (b) a retroviral-derived packaging signal sequence (ψ), (c) a sequence derived from a nucleic acid encoding a gag protein having a length of 183 to 227 bp, (d) a desired sequence or a multicloning site, and (e) a retroviral-derived 3' LTR sequence. [2] The nucleic acid construct of [1], further comprising a post-transcriptional regulatory element (PRE) into which a sequence that causes a frameshift in the nucleic acid sequence encoding the X protein or a stop codon that interrupts translation of the X protein has been inserted. [3] The nucleic acid construct of [2], wherein the PRE is a woodchuck hepatitis virus-derived PRE (WPRE). [4] The nucleic acid construct of [1], wherein the exogenous promoter sequence is a cytomegalovirus-derived promoter. [5] The nucleic acid construct according to [1], wherein the desired sequence is a sequence comprising an internal promoter sequence. [6] The nucleic acid construct according to [1], wherein the desired sequence comprises a sequence encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR). [7] The nucleic acid construct according to [6], wherein the desired sequence comprises a sequence encoding a TCRα chain and a TCRβ chain linked by a 2A peptide. [8] The nucleic acid construct according to [1], wherein the LTR sequence is a sequence derived from a lentivirus. [9] The nucleic acid construct according to [1], wherein the 3'LTR sequence is a self-inactivating (SIN) LTR sequence.

[10] A retroviral vector comprising a transcript from the nucleic acid construct according to any one of [1] to [9].

[11] The retroviral vector according to

[10] , wherein the 5'LTR, packaging signal sequence, and 3'LTR derived from an oncoretrovirus or a lentivirus.

[12] A method for producing a retroviral vector, comprising the step of introducing the nucleic acid construct according to any one of [1] to [9] into a cell capable of producing retroviral particles.

[13] A method for producing a gene-transduced cell, comprising the step of introducing the retroviral vector according to

[10] or

[11] into a cell.

[14] The method for producing transgenic cells according to

[13] , wherein the cells are immune cells, cells that can differentiate into immune cells, or cell populations containing them.

[0010] The present invention provides a highly safe nucleic acid construct for producing a retrovirus, which can be used to efficiently express a desired gene, a retroviral vector containing a transcription product from the nucleic acid construct, a method for producing a gene-transduced cell using the vector, and cells containing the nucleic acid construct. These nucleic acid constructs, retroviral vectors, and cells are extremely useful for protein production, disease treatment by cell therapy, and research and testing related to such treatments.

[0011] FIG. 1 shows the structure of a nucleic acid construct prepared in an example. FIG. 2 shows the relative values ​​of fluorescence intensity of ZsGreen1 protein expressed by cells infected with retroviral vectors produced using each nucleic acid construct. FIG. 3 shows the relative values ​​of virus titer of retroviral solutions produced using each nucleic acid construct. FIG. 4 shows the structure of a nucleic acid construct prepared in an example. FIG. 5 shows the virus titer of retroviral solutions produced using each nucleic acid construct. FIG. 6 shows the structure of a nucleic acid construct prepared in an example. FIG. 7 shows the virus titer of retroviral solutions produced using each nucleic acid construct. FIG. 8 shows the percentage of cells positive for CAR protein expression after infection with retroviral vectors produced using each nucleic acid construct. FIG. 9 shows the number of virus copies incorporated into the genome of cells infected with retroviral vectors produced using each nucleic acid construct. FIG. 10 shows the cytotoxic activity of CAR-expressing cells infected with retroviral vectors produced using each nucleic acid construct. FIG. 11 shows the structure of a nucleic acid construct prepared in an example. FIG. 12 shows the RNA expression level of wild-type TCR protein expressed by cells infected with retroviral vectors produced using each nucleic acid construct. FIG. 1 shows the RNA expression levels of codon-modified TCR proteins expressed by cells infected with retroviral vectors produced using each nucleic acid construct.

[0012] As used herein, the term "nucleic acid construct" refers to a nucleic acid comprising a sequence constructed to include one or more functional units not found in nature. The nucleic acid may be DNA and / or RNA and may also include modified nucleic acids. Examples of such forms include circular, linear, double-stranded, single-stranded, extrachromosomal DNA molecules (plasmids), and cosmids. Furthermore, a nucleic acid construct may contain, in addition to a nucleic acid sequence encoding a gene, a nucleic acid sequence containing a control sequence (e.g., a promoter) operably linked (i.e., capable of controlling transcription and translation) as needed. The nucleic acid construct may also contain other regulatory elements, functional sequences, linkers, and the like as needed.

[0013] As used herein, "LTR (Long Terminal Repeat)" refers to a sequence consisting of 100 to 1000 base pairs that is repeated at both ends of proviral DNA such as retroviruses and retrotransposons. LTR is composed of U3, R, and U5 regions that are involved in transcription of the viral genome, reverse transcription from the viral genome, and integration of double-stranded DNA synthesized through reverse transcription into host DNA. The IR sequences (inverted repeat regions) at the 5' and 3' ends of the provirus are 4 to 20 base pairs in length. U3 includes a transcription enhancer sequence and a promoter sequence.

[0014] As used herein, the term "packaging signal sequence," also referred to as "psi sequence" or "ψ sequence," refers to a non-coding cis-acting sequence required for encapsidation of a retroviral RNA strand and packaging into viral particles during viral particle formation. For example, the region from the 3' side of the major splice donor (SD) site to the gag initiation codon, or the region from the 3' side of the SD site to a portion of the gag gene sequence, is used.

[0015] As used herein, the term "desired sequence of interest" refers to an exogenous sequence that is desired to be artificially (by artificial manipulation) inserted into a cell (e.g., the nuclear genome or cytoplasm of a cell) either transiently or permanently. Such sequences include gene sequences that are completely or partially heterologous to the cell into which they are introduced, and also include gene sequences that contain any mutations. They may also be gene sequences identical to endogenous genes naturally present in the cell. Here, "naturally" refers to a natural state that has not been artificially manipulated.

[0016] As used herein, a "multi-cloning site" refers to a cluster sequence consisting of multiple restriction enzyme sites for cloning. There are no particular limitations on the base sequence constituting the multi-cloning site or the type and number of restriction enzyme sites included.

[0017] As used herein, the term "posttranscriptional regulatory element (PRE)" refers to a sequence that contributes to promoting polyadenylation of mRNA transcribed from a gene in a cell, promoting the nuclear export of mRNA, or activating the translation of mRNA. By inserting a PRE into the untranslated region of a desired gene contained in the nucleic acid construct of the present invention, the expression of the desired gene at the protein level is improved.

[0018] As used herein, "wild-type" refers to a gene or gene product isolated from a naturally occurring source that is most frequently observed in a population. It can be isolated from nature or artificially created. "Mutant," on the other hand, refers to a gene or gene product that has altered sequence and / or functional properties when compared to a wild-type gene or gene product. Mutant genes are produced by natural mutations or by artificially modifying a gene to alter its sequence.

[0019] As used herein, "T cells," also known as T lymphocytes, refer to thymus-derived lymphocytes involved in immune responses. Examples of T cells include helper T cells, suppressor T cells, regulatory T cells, CTLs, naive T cells, memory T cells, αβ T cells expressing α- and β-chain TCRs, and γδ T cells expressing γ- and δ-chain TCRs. "Cells capable of differentiating into T cells" are not particularly limited as long as they differentiate into T cells in vivo or upon artificial stimulation, and include, for example, hematopoietic stem cells, multipotent progenitor cells, common lymphoid progenitor cells, and T cell precursor cells. Examples of "cell populations containing T cells or cells capable of differentiating into T cells" include blood (peripheral blood, umbilical cord blood, etc.), bone marrow fluid, and cell populations containing peripheral blood mononuclear cells (PBMCs), blood cells, hematopoietic stem cells, and cord blood mononuclear cells collected, isolated, purified, or induced from these blood cells. Furthermore, various cell populations derived from blood cells containing T cells can be used in the present invention. These cells may be activated in vivo or ex vivo with an anti-CD3 antibody or cytokines such as IL-2. These cells may be collected from a living body or obtained by ex vivo culture. For example, T cell populations obtained from a living body may be used either directly or after cryopreservation.

[0020] The present invention will be described in detail below. (1) Nucleic Acid Construct of the Present Invention The nucleic acid construct of the present invention is a nucleic acid construct for producing a retroviral vector, comprising, from the 5' end, the following sequences: (a) a retroviral-derived 5' LTR (Long Terminal Repeat) sequence containing an exogenous promoter sequence; (b) a retroviral packaging signal sequence (ψ); (c) a sequence derived from a nucleic acid encoding a gag protein having a length of 183 to 227 bp; (d) a desired sequence or a multicloning site; and (e) a retroviral-derived 3' LTR sequence. The nucleic acid construct of the present invention can be used to produce a retroviral vector. That is, by introducing the nucleic acid construct of the present invention into cells capable of producing retroviral particles, a retroviral vector of the present invention containing a transcript from the nucleic acid construct can be produced. The nucleic acid construct of the present invention enables the production of a retroviral vector with a high viral titer. This retroviral vector makes it possible to introduce a desired sequence into cells. Cells into which the desired sequence has been introduced by the retroviral vector of the present invention exhibit high expression efficiency of the desired sequence.

[0021] Retroviruses are enveloped viruses with a genome of positive-sense single-stranded RNA, and the major elements of the viral genome, from the 5' end, are the 5' LTR sequence, SD sequence, packaging signal sequence, gag gene, pol gene, SA sequence, env gene, and 3' LTR sequence. In the case of lentiviruses, which will be described later, multiple accessory genes are contained in addition to these elements. Of these, the 5' LTR sequence, packaging signal sequence, and 3' LTR sequence are essential for retroviral vectors in gene transfer systems using retroviral vectors, and the nucleic acids of the present invention possess all of these. Other gene products, such as gag, pol, and env, can be supplied by packaging cells harboring these genes. Typically, the desired sequence is located 3' to the packaging signal sequence of the retroviral vector, or, if an SA sequence is present, 3' to the packaging signal sequence and SA sequence.

[0022] The nucleic acid constructs of the present invention comprise a sequence derived from a nucleic acid encoding a gag protein, which is 183 to 227 bp in length. While retroviral packaging signal sequences cannot be completely removed because they partially contain the gag gene sequence, a sequence of this length is useful for efficient virus production and expression of a desired sequence. Furthermore, the present invention can reduce the risk of homologous recombination with the full-length gag gene used in virus production, thereby providing a highly safe method for producing a viral vector. For example, a sequence derived from a nucleic acid encoding a gag protein is preferably 183 bp or 227 bp in length, and examples thereof include the sequence set forth in SEQ ID NO: 4 or 3, or a base sequence in which one or several, e.g., one to nine, bases have been substituted, deleted, inserted, or added to this sequence.

[0023] The (a) 5' LTR sequence containing an exogenous promoter sequence, (e) 3' LTR sequence, and (b) packaging signal sequence contained in the nucleic acid construct of the present invention are sequences derived from retroviruses, and any sequences that enable the production of a retrovirus containing RNA comprising these sequences as its genome can be used. Retroviruses include the subclasses of oncoretroviruses and lentiviruses, and sequences derived from viruses of either class can be used in the present invention. These sequences may be derived from the same virus, or sequences derived from different viruses may be used in combination as long as they allow the formation of viral particles and integration into the genome of the transduced cell when combined with appropriate packaging cells.

[0024] The LTR sequence and packaging signal sequence used in the present invention may be derived from, for example, Moloney murine leukemia virus (MMLV), mouse embryonic stem cell virus (MESV), murine stem cell virus (MSCV), myeloproliferative sarcoma virus (MPSV), or spleen-limited focus-forming virus (SFFV), which belong to the oncoretroviruses. Viral vectors derived from oncoretroviruses are capable of highly efficient gene transfer, but require that cells are actively dividing at the time of vector transfer. These oncoretroviral vectors have been described in numerous publications [e.g., U.S. Pat. Nos. 5,219,740, 6,207,453, 5,219,740, BioTechniques, Vol. 7, pp. 980-990 (1989), Human Gene Therapy, Vol. 1, pp. 5-14 (1990), Virology, Vol. 180, pp. 849-852 (1991), Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 8033-8037 (1993), and Current Opinion in Genetics and Cur. Opin. Genet. Develop., vol. 3, pp. 102-109 (1993)].

[0025] Furthermore, the LTR sequences and packaging signal sequences used in the present invention may be derived from, for example, lentiviruses such as human immunodeficiency viruses (HIV-1, HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), and caprine arthritis-encephalitis virus (CAEV). Lentiviral vectors can transfer genes into the nuclear genome of cells regardless of mitosis. Lentiviral vectors have also been described in numerous publications [e.g., J. Virology, Vol. 72, pp. 8463-8471 (1998)]. Other retrovirus groups, such as the spumavirus genus (e.g., foamy viruses), can also efficiently transduce non-dividing cells.

[0026] The LTR is functionally divided into three regions, U3, R, and U5, from the 5' end. The U3 region has enhancer / promoter activity, and the viral genome is transcribed from the R region of the 5'LTR sequence to the R region of the 3'LTR sequence by host cell RNA polymerase II. The 5'LTR sequence used in the present invention contains an enhancer / promoter exogenous to the virus from which the LTR is derived. In one embodiment of the present invention, the U3 region of the 5'LTR is replaced with an exogenous enhancer / promoter. Furthermore, LTR sequences in which the U3 region of the 3'LTR sequence is replaced with an enhancer / promoter derived from a source other than the virus from which the LTR sequence is derived can also be used in the present invention. The exogenous enhancer / promoter to be replaced can be a sequence derived from a virus or a mammal, and a constitutive, inducible, or tissue-specific enhancer / promoter can be used. For example, enhancers / promoters derived from viruses such as human cytomegalovirus (HCMV) immediate early, Moloney murine sarcoma virus (MMSV), murine stem cell virus (MSCV), Rous sarcoma virus (RSV), and spleen focus-forming virus (SFFV), as well as mammalian enhancers / promoters for β-actin, globin, elastase, albumin, α-fetoprotein, and insulin genes, can be used. Here, the term "enhancer / promoter" refers to a sequence containing an enhancer region and / or a promoter region. Generally, enhancer regions and promoter regions are sometimes collectively referred to as "promoters." Furthermore, to distinguish them from enhancer regions, promoter regions are sometimes referred to as "core promoters." Either enhancer regions and / or promoter regions can be used in the present invention. In one embodiment of the present invention, the 5' LTR sequence containing an exogenous promoter sequence comprises the sequence set forth in SEQ ID NO: 7, or a nucleotide sequence in which one or several, for example, 1 to 9, bases have been substituted, deleted, inserted, or added to this sequence.

[0027] The 3'LTR sequence used in the present invention can be a sequence in which enhancer / promoter activity has been deleted by introducing a mutation into the U3 region. As a result, when a viral vector containing a transcript from this nucleic acid construct infects a cell, transcription from the R region is suppressed in the provirus formed when the viral genome is integrated into the cellular chromosome. Retroviral vectors in which the U3 region of the 3'LTR sequence has been mutated in this way are called self-inactivating (SIN) vectors. The introduction of a mutation into the 3'LTR sequence is achieved by base substitution or deletion. Because SIN vectors suppress transcription from the R region of the LTR in the provirus, a promoter sequence for expressing the desired sequence must be located separately from the LTR. Furthermore, when expressing multiple desired sequences, a promoter sequence separate from the LTR is also located. Such a promoter sequence located between the 5'LTR and 3'LTR is sometimes referred to as an internal promoter sequence. Promoter sequences derived from viruses or mammalian genes can be used as internal promoter sequences. When a virus-derived promoter sequence is used, it can be derived from the same virus as the virus from which the 5' LTR, 3' LTR, or packaging signal sequence is derived, or from a different virus. For example, a promoter sequence derived from the U3 region of a retroviral LTR, such as a promoter sequence derived from the U3 region of the LTR of murine stem cell virus (MSCV), can be used. Furthermore, the internal promoter sequence can be any of the exogenous promoter sequences exemplified in the previous paragraph that replace the U3 region of the 5' LTR sequence. In addition to the above, virus-derived promoter sequences such as the SV40 promoter and CMV promoter can also be used. Furthermore, promoter sequences that function in mammalian cells, such as the phosphoglycerate kinase (PGK) promoter, polypeptide chain elongation factor (EF1-α) promoter, β-actin promoter, and CAG promoter, can also be used. The internal promoter may be located upstream (i.e., 5'-side) of (d), between (a) and (d), preferably between (b) and (d).

[0028] The nucleic acid construct of the present invention may contain an SD sequence and / or an SA sequence. These sequences may be exogenous SD sequences and / or SA sequences relative to the LTR or exogenous SD sequences and / or SA sequences relative to the internal promoter sequence. The SD sequence and SA sequence may also be derived from different sources. For example, SD sequences and SA sequences derived from simian virus (SV) 40 16S RNA, HCMV immediate early RNA, or the human hEF1α gene may be used [Proceedings of the National Academy of Sciences of the United States of America, Vol. 95, No. 1, pp. 219-223 (1998)]. Furthermore, SD sequences or SA sequences with enhanced or suppressed splicing activity due to mutations introduced into the consensus sequence may also be used in the nucleic acid construct of the present invention.

[0029] The desired sequence (d) contained in the nucleic acid construct of the present invention is a sequence desired to be expressed in cells into which the produced vector is introduced. Examples of such sequences include protein-encoding sequences and sequences encoding RNAs that function within cells, such as tRNA and miRNA. Alternatively, a nucleic acid construct may be produced in which a sequence (d) containing multiple restriction enzyme recognition sequences for ligating the desired sequence (multi-cloning site) is arranged, and the desired sequence may then be inserted using the multi-cloning site. Such nucleic acid constructs having a multi-cloning site instead of the desired sequence are also included in the nucleic acid constructs of the present invention.

[0030] The desired sequence may be intended for the prevention or treatment of a disease. Examples of such a sequence include sequences useful for suppressing the transcription or expression of harmful gene products in a living body (e.g., sequences encoding siRNA), sequences encoding proteins for supplementing proteins that are missing or have lost their function in a living body, and sequences that can modify or enhance cellular functions. The present invention provides gene therapy in which cells containing a foreign sequence introduced into a living body using the nucleic acid construct of the present invention are introduced into the body. Examples of such gene therapy include those using a sequence encoding the IL-2 receptor γ chain (X-linked severe combined immunodeficiency), a sequence encoding β-globin (β-thalassemia), a sequence encoding adenosine deaminase (ADA) (ADA deficiency), a sequence encoding a blood coagulation factor (hemophilia), or a sequence encoding an antigen-recognizing receptor (cancer or viral infection).

[0031] In one embodiment of the present invention, the desired sequence contained in the nucleic acid construct of the present invention is a sequence encoding an oligomeric protein. Oligomeric proteins include structural proteins, enzymes, transcription factors, receptors, and antibodies. In addition, in the present invention, the oligomeric protein may be a cell surface protein (membrane protein), and a sequence encoding an antigen-recognition receptor, such as a T cell receptor (TCR), as exemplified in the Examples is suitable as the desired sequence.

[0032] In one embodiment of the present invention, the desired sequence contained in the nucleic acid construct of the present invention is a sequence encoding a chimeric antigen receptor (CAR). A typical CAR structure comprises a single-chain antibody (single chain variable fragment: scFv) that recognizes a surface antigen on target cells to be eliminated from the body, such as tumor cells, a transmembrane domain, and an intracellular domain that activates T cells. The intracellular domain of the TCR complex CD3ζ is preferably used as the intracellular domain. A CAR with such a configuration is called a first-generation CAR. The gene encoding the single-chain antibody portion can be isolated, for example, from a hybridoma that produces a monoclonal antibody that recognizes the target antigen. T cells expressing the CAR directly recognize surface antigens on target cells, regardless of the expression of major histocompatibility complex class I on tumor cells, and simultaneously activate T cells, thereby enabling efficient killing of the target cells.

[0033] Second-generation CARs have been developed in which the intracellular domain of a T cell costimulatory molecule is linked to enhance the T cell activation ability of first-generation CARs. The intracellular domains of CD28, CD137 (4-1BB), or CD134 (OX40), which belong to the tumor necrosis factor (TNF) receptor superfamily, are preferably used as T cell costimulatory molecules. As further improved versions, third-generation CARs have also been developed in which the intracellular domains of these costimulatory molecules are linked in tandem, and many CAR molecules targeting various tumor antigens have been reported. The nucleic acid construct of the present invention may comprise, as a desired sequence, a sequence encoding any of the CARs.

[0034] The nucleic acid construct of the present invention may contain a posttranscriptional regulatory element (PRE), which is useful for enhancing expression of a desired sequence. The PRE is located within an intron of the transcript from the nucleic acid construct and can be removed by splicing during the retroviral life cycle. Examples of PREs that are not removed by splicing include the posttranscriptional processing element of herpes simplex virus, the posttranscriptional regulatory elements of hepatitis B virus (HPRE), and woodchuck hepatitis virus (WPRE). The PRE sequence includes the coding region of X protein, which has been suggested to be carcinogenic. The nucleic acid construct of the present invention may also use a mutant sequence that suppresses X protein expression from the PRE sequence. For example, a sequence that causes a frameshift in the nucleic acid sequence encoding X protein or a PRE sequence into which a stop codon that interrupts X protein translation is inserted may be used. In one embodiment of the present invention, a PRE sequence containing one or two inserted bases that causes a frameshift between positions 6 and 7, with A of the start codon ATG of X protein as position 1, may be used. Alternatively, a PRE sequence in which positions 7 to 9 have been substituted with a stop codon (e.g., TAA) can be used. WPRE is preferred for the present invention. For details of WPRE, see U.S. Pat. No. 6,136,597 or U.S. Pat. No. 7,419,829. In one embodiment of the present invention, WPRE2 (SEQ ID NO: 8) or WPRE3 (SEQ ID NO: 9) or a base sequence in which one or several, for example, 1 to 9 bases have been substituted, deleted, inserted, or added to this sequence can be used. One embodiment of the present invention is exemplified by a nucleic acid construct in which a PRE sequence is located between (d) a desired sequence or a multicloning site and (e) a 3' LTR sequence derived from a retrovirus.

[0035] The nucleic acid construct of the present invention may contain a Rev response element (RRE) sequence and / or a central polypurine tract (cPPT) sequence. Examples of RRE include, but are not limited to, the RRE located at positions 7622 to 8459 of the HIV NL4-3 genome (GenBank Accession No. AF003887), and RREs derived from other strains of HIV or other retroviruses. Furthermore, the cPPT is a sequence of approximately 15 bases located approximately in the center of the lentiviral genome, and serves as a primer binding site for plus-strand DNA synthesis during double-stranded DNA synthesis from the lentiviral genomic RNA. When the lentiviral RNA genome is reverse transcribed, the cPPT functions together with the central termination sequence (CTS) to form a triple-stranded structure called a DNA flap. In the nucleic acid construct of the present invention, the arrangement of these sequences is not particularly limited, and they may be arranged in either order from the 5' end: cPPT, RRE, or RRE, cPPT.

[0036] The present invention includes a method for producing retroviral vectors (particles), comprising the step of introducing the nucleic acid construct of the present invention into cells capable of producing retroviral particles. The nucleic acid construct can be introduced into cells using an appropriate vector, such as a plasmid vector, a non-retroviral virus vector, or a transposon vector, so that it can stably exert its effect within the cells. Furthermore, it can be integrated into the chromosomal DNA of the cells using genome editing techniques. Alternatively, the nucleic acid construct can be directly introduced into cells. To introduce the nucleic acid construct into cells directly or using a plasmid vector, methods using carriers such as liposomes or ligand-polylysine, calcium phosphate methods, electroporation, particle gun methods, and the like can be used. The viral vector is not particularly limited, and commonly used viral vectors used in gene transfer methods, such as adenoviral vectors, adeno-associated viral vectors, simian viral vectors, vaccinia viral vectors, measles viral vectors, or Sendai viral vectors, are commonly used.

[0037] Known packaging cells can be used as cells capable of producing retroviral particles. Appropriate packaging cells can be selected based on the LTR sequence and packaging signal sequence contained in the nucleic acid construct, and the nucleic acid construct can be introduced into retrovirus-producing cells to prepare retroviral vectors (particles). Alternatively, the nucleic acid construct of the present invention and nucleic acids encoding components necessary for retroviral particle production (gag gene, pol gene, env gene, and other accessory genes) can be simultaneously or sequentially introduced into cells with high transfection efficiency (e.g., 293 cells or 293T cells), and these cells can be cultured in an appropriate medium as retroviral-producing cells to produce retroviral particles. The produced retroviral particles contain transcripts from the nucleic acid construct of the present invention. These retroviral particles are included in the present invention as retroviral vectors for introducing desired sequences into cells. Examples of packaging cells include PG13 (ATCC CRL-10686) and PA317 (ATCC CRL-9078). Packaging cells and other cells, kits containing plasmids for producing retroviruses (called packaging plasmids), and the like are widely available commercially from various companies, and these can be used in the methods of the present invention.

[0038] In the present invention, pseudotyped retroviruses can be produced by using packaging cells expressing an envelope protein derived from a virus heterologous to that from which the genome of the retroviral vector is derived, or by using a packaging plasmid containing a sequence encoding the envelope protein. For example, packaging plasmids encoding envelopes derived from MMLV, gibbon ape leukemia virus (GaLV), vesicular stomatitis virus (VSV), or feline endogenous virus, or proteins capable of functioning as an envelope, can be used. Furthermore, retroviral vectors bearing glycosylated proteins on their surface can be produced by using packaging cells into which genes for enzymes involved in glycosylation have been introduced.

[0039] After culturing the retrovirus-producing cells prepared by the above procedure, the cell culture is centrifuged to collect the supernatant, and contaminants are removed by appropriate filtration to obtain retrovirus particles. These crudely purified retrovirus particles can be directly contacted with cells for gene transfer, but higher purity retrovirus particles can also be prepared by known purification procedures and then subjected to gene transfer.

[0040] The present invention provides a composition comprising a retroviral vector of the present invention as an active ingredient together with a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are well known to those skilled in the art and include, for example, phosphate-buffered saline (e.g., 0.01 M phosphate, 0.138 M NaCl, 0.0027 M KCl, pH 7.4), aqueous solutions containing mineral acid salts such as hydrochloride, hydrobromide, phosphate, and sulfate, physiological saline, glycol or ethanol solutions, and salts of organic acids such as acetate, propionate, malonate, and benzoate. Auxiliaries such as wetting agents or emulsifiers, and pH buffers may also be used. Pharmaceutically acceptable excipients include those described in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991) (incorporated herein by reference). The composition can be in a known form suitable for parenteral administration, for example, injection or infusion. In addition, formulation aids such as suspending agents, preservatives, stabilizers and / or dispersing agents, and preservatives for extending shelf life during storage can be used. The composition can also be in a dry form for reconstitution with an appropriate sterile liquid before use.

[0041] (2) Method for Producing Genetically Induced Cells of the Present Invention The method for producing gene-inducing cells of the present invention is characterized by comprising the step of introducing into cells a retroviral vector comprising the nucleic acid construct of the present invention described in (1) above or a transcript from the nucleic acid construct. DNA corresponding to the region sandwiched between the 5' LTR and 3' LTR of the nucleic acid construct of the present invention is integrated into the chromosome of the gene-inducing cell of the present invention. In one embodiment of the present invention, this step is carried out ex vivo.

[0042] The methods of the present invention can use cells derived from mammals, such as humans, or cells derived from non-human mammals, such as monkeys, mice, rats, pigs, cows, and dogs. The cells used in the methods of the present invention are not particularly limited, and any cells can be used. For example, cells collected, isolated, purified, or induced from body fluids, tissues, or organs, such as blood (peripheral blood, umbilical cord blood, etc.), bone marrow, or other body fluids can be used. Peripheral blood mononuclear cells (PBMCs), immune cells [T cells, dendritic cells, B cells, hematopoietic stem cells, macrophages, monocytes, NK cells, or blood cells (neutrophils, basophils)], umbilical cord blood mononuclear cells, fibroblasts, preadipocytes, hepatocytes, skin keratinocytes, mesenchymal stem cells, adipose stem cells, various cancer cell lines, or neural stem cells can be used. In the present invention, immune cells, immune cell precursors (hematopoietic stem cells, lymphocyte precursor cells, etc.), or cell populations containing these cells are particularly preferred. T cells, which are representative of immune system cells, include αβ T cells, γδ T cells, CD8+ T cells, CD4+ T cells, regulatory T cells, cytotoxic T cells, and tumor-infiltrating lymphocytes. Cell populations containing T cells and T cell precursors include PBMCs. Furthermore, NK cells, NKT cells, and their precursors can also be used in the methods of the present invention. Examples of the above cells include, but are not limited to, those collected from a living organism, those expanded therefrom, those established as cell lines, and those differentiated from pluripotent stem cells. When it is desired to transplant the produced transgenic cells or cells differentiated from such cells into a living organism, it is preferable to produce the transgenic cells from cells collected from the living organism itself or from an allogeneic organism.

[0043] In the step of introducing the retroviral vector of the present invention into cells, functional substances that improve the efficiency of introduction can also be used (e.g., WO 95 / 26200, WO 00 / 01836). Substances that improve the efficiency of introduction include substances that have the activity of binding to viral vectors, such as fibronectin or fibronectin fragments. Preferably, a fibronectin fragment having a heparin-binding site can be used, such as a fragment commercially available as RetroNectin (RetroNectin, registered trademark, CH-296, manufactured by Takara Bio Inc.). Commercially available adjuvants for retroviral gene introduction can also be used.

[0044] In a preferred embodiment of the present invention, the functional substances may be used in a manner appropriate for each substance. For example, in the case of retronectin, it can be used in a state immobilized on an appropriate solid phase, such as a container used in cell culture (a plate, a petri dish, a flask, a bag, etc.) or a carrier (microbeads, etc.).

[0045] (3) Cells of the Present Invention The cells of the present invention are transgenic cells produced by the production method of (2) above. The cells of the present invention express a gene product encoded by an exogenous desired sequence. Therefore, the transgenic cells of the present invention have acquired new properties and / or functions attributable to the gene product.

[0046] In one embodiment of the present invention, the cells of the present invention can be used as a therapeutic agent for a disease. The therapeutic agent contains, as an active ingredient, the cells of the present invention capable of expressing a gene product useful for treating a disease, and may further contain an appropriate excipient. The excipient is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include stabilizers, buffers, and isotonicity agents. The diseases to which the cells of the present invention are administered are not particularly limited as long as they are sensitive to the cells, and examples thereof include cancers [blood cancers (leukemia), solid tumors, etc.], inflammatory diseases / autoimmune diseases (asthma, eczema, etc.), hepatitis, and infectious diseases caused by viruses, bacteria, or fungi (influenza, AIDS, tuberculosis, MRSA infection, VRE infection, deep mycosis). Cells expressing TCRs or CARs that recognize antigens possessed by cells whose reduction or elimination is desired in the above-mentioned diseases, i.e., tumor antigens, viral antigens, bacterial antigens, etc., are administered to treat these diseases. The cells of the present invention can also be used for preventing infectious diseases after bone marrow transplantation or radiation exposure, and for donor lymphocyte infusion for the purpose of remission of relapsed leukemia. Therapeutic agents comprising the cells of the present invention as an active ingredient can be administered parenterally, for example, by injection or infusion, intradermally, intramuscularly, subcutaneously, intraperitoneally, intranasally, intraarterially, intravenously, intratumorally, or into afferent lymphatic vessels, but are not limited thereto.

[0047] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Furthermore, among the procedures described herein, the basic procedures were performed in accordance with the method described in Molecular Cloning: A Laboratory Manual, 3rd ed., edited by T. Maniatis et al., published by Cold Spring Harbor Laboratory in 2001.

[0048] Example 1: Preparation of Gag Residual Sequence-Reduced Virus Vector Plasmid First, a DNA fragment of the MSCV U3 promoter sequence shown in SEQ ID NO: 1 was amplified by PCR using pMSCVneo (Clontech) as a template. Next, a DNA fragment of the ZsGreen1 sequence shown in SEQ ID NO: 2 was amplified by PCR using pLVSIN-IRES-ZsGreen1Vector as a template. These DNA fragments were inserted into the ClaI-MluI digestion product of pLVSIN-CMV-Neo (Takara Bio Inc.) to prepare the pLVSIN-MSCV-ZsGreen1 plasmid.

[0049] Next, as shown in Figure 1, the sequence derived from the HIV-1 Gag sequence contained in pLVSIN-MSCV-ZsGreen1 was deleted by 133 bp, 177 bp, 222 bp, 267 bp, and 178 bp (45 + 133), respectively, to prepare Vectors X, V, W, Y, and Z. The sequences derived from the HIV-1 Gag sequence contained in Gag sequence-deleted Vectors X, V, W, and Y are set forth in SEQ ID NOs: 3, 4, 5, and 6, respectively.

[0050] Example 2 Preparation of Retrovirus Solutions Escherichia coli JM109 was transformed with pLVSIN-MSCV-ZsGreen1 prepared in Example 1 and vectors X, V, W, Y, and Z. Plasmid DNA contained in these transformants was purified using NucleoSpin (registered trademark) Plasmid Midi (Machlinergel GmbH) and subjected to the following procedure as transfection DNA. Each of the prepared plasmids and a packaging plasmid (transiently expressing HIV-1-derived lentiviral proteins Gag, Pol, Tat, and Rev, as well as VSV-G envelope protein) were introduced into 293T cells (ATCC CRL-11268), and the resulting cells were cultured to prepare supernatants containing lentiviruses bearing the VSV-G envelope. The supernatants were filtered through a 0.45 μm filter (Milex HV, manufactured by Millipore) to give virus solutions LVSIN-MSCV-ZsGreen1, solution X, solution Y, solution V, solution W, and solution Z, respectively.

[0051] Example 3: Infection with Gag Sequence-Reduced Lentiviral Vector The virus solution prepared in Example 2 was appropriately diluted and used to infect SupT1 cells (ATCC CRL-1942), a cell line derived from human T lymphocytic leukemia, once. Using a flow cytometer, the proportion of ZsGreen1-positive cells and the mean fluorescence intensity among the positive cells were measured 3 and 4 days after virus infection, and the virus titer was calculated according to the following formula.

[0052] Virus titer (IFU / mL) = number of infected cells × (positive rate % / 100) × virus dilution ratio / volume of solution at infection (mL)

[0053] Figure 2 shows a comparison of the mean fluorescence intensity, with the vector LVSIN-MSCV-ZsGreen1 set as 100%, and Figure 3 shows a comparison of the virus titers. In both cases, the average of triplicate test values ​​was calculated. As shown in Figure 2, vectors X, V, W, and Y showed mean fluorescence intensity equivalent to that of vector LVSIN-MSCV-ZsGreen1. As shown in Figure 3, a significant decrease in virus titer was observed for vectors W and Y, while no significant decrease was observed for vectors X and V.

[0054] Example 4 Hybrid LTR and WPRE2 pLVSIN-EF1α-ZsGreen1 was prepared by replacing the MSCV-U3 promoter sequence of pLVSIN-MSCV-ZsGreen1 prepared in Example 1 with a human EF1α promoter sequence. Furthermore, the 5'LTR sequences of the two vectors were replaced with an LTR sequence (SEQ ID NO: 7) containing a CMV promoter sequence as a foreign promoter sequence to create a hybrid LTR, and 177 bp of the remaining GAG sequence was deleted to create a 183 bp sequence (SEQ ID NO: 4). Furthermore, the WPRE sequence was replaced with WPRE2 (SEQ ID NO: 8), which had an inserted base pair that caused a frameshift between positions 6 and 7 of the ATG initiation codon of X protein, with the A at position 1, to suppress expression of the X protein contained in the sequence. In this way, pLGT2-MSCV-ZsGreen1 and pLGT2-EF1α-ZsGreen1 were constructed. The structures of each vector are shown in FIG.

[0055] Escherichia coli HST08 was transformed with each of the prepared plasmid DNAs. The plasmid DNA contained in these transformants was purified using NucleoSpin (registered trademark) Plasmid Midi (Machlinergel) and subjected to the following procedure as transfection DNA. Each of the prepared plasmid DNAs and the packaging plasmid used in Example 2 were transfected into 293T cells, and four types of supernatants containing lentiviruses with a VSVG envelope were obtained. Note that pLGT2-MSCV-ZsGreen1 and pLGT2-EF1α-ZsGreen1 used packaging plasmids that did not contain a plasmid expressing the lentiviral TAT protein. The supernatant was filtered through a 0.45 μm filter (Milex HV, Millipore) to prepare virus solutions, designated LVSIN-MSCV, LVSIN-EF1α, LGT2-MSCV, and LGT2-EF1α.

[0056] The prepared virus solution was infected into J45.01 cells (ATCC CRL-1990) at various dilutions. Four days after virus infection, the cells were harvested, and the percentage of cells expressing the ZsGreen1 gene was measured using a flow cytometer. The virus titer was calculated using the measured value at which the ZsGreen1 positivity rate was 1.0-20.0%, and the results are shown in Figure 5. As shown in Figure 5, equivalent virus titers were obtained for all lentiviral vectors.

[0057] Furthermore, to suppress expression of the X protein contained in the WPRE sequence of pLGT2-MSCV-ZsGreen1 and pLGT2-EF1α-ZsGreen1, a plasmid DNA containing WPRE3 (SEQ ID NO: 9) was prepared in which the A of the start codon ATG of the X protein was replaced with a stop codon (TAA) at positions 7 to 9, with the A at position 1. Virus solutions were prepared in the same manner as above, and J45.01 cells were infected to confirm that equivalent virus titers were obtained.

[0058] Regarding the expression of the X protein contained in the WPRE sequence, a sequence encoding the fluorescent protein AcGFP was linked to the 3' end of each of the wild-type WPRE sequence, WPRE2, and WPRE3, and the expression of a fusion protein of the X protein and AcGFP was confirmed. Measured values ​​of fluorescence intensity indicated that the X protein was not expressed from WPRE2 and WEPRE3, confirming the high safety of WPRE2 and WEPRE3.

[0059] Example 5 Expression of CAR Gene Plasmid DNA was prepared by replacing the ZsGreen1 sequence of each vector plasmid DNA prepared in Example 4 with a sequence (MSLN-CAR) encoding a chimeric antigen receptor (CAR) that specifically recognizes mesothelin (MSLN). These plasmid DNAs were named pLVSIN-MSCV-MSLN-CAR, pLVSIN-EF1α-MSLN-CAR, pLGT2-MSCV-MSLN-CAR, and pLGT2-EF1α-MSLN-CAR, respectively. The structure of each vector is shown in Figure 6.

[0060] Using the prepared plasmids, virus solutions named LVSIN-MSCV, LVSIN-EF1α, LGT2-MSCV, and LGT2-EF1α were prepared in the same manner as in Example 4. The virus titers were measured in the same manner as in Example 4, and the results are shown in Figure 7. As shown in Figure 7, the lentiviral vectors prepared from plasmid DNA containing hybrid LTRs exhibited virus titers equal to or higher than those of conventional lentiviral vectors.

[0061] Example 6: Function of CAR gene-transfected cells Using the plasmid DNA containing the hybrid LTR prepared in Example 5, peripheral blood mononuclear cells (PBMCs) isolated from human peripheral blood were infected with virus solutions LGT2-MSCV and LGT2-EF1α containing the MSLN-CAR sequence at various dilutions using RetroNectin (Takara Bio Inc.). Seven days after virus infection, the cells were harvested and stained with a biotin-labeled anti-mouse IgG antibody (Jackson ImmunoResearch) and an FITC-labeled anti-human CD8 antibody (Becton Dickinson). The percentage of cells positive for anti-MSLN-CAR expression was measured using a flow cytometer. The results are shown in Figure 8. As shown in Figure 8, it was confirmed that the anti-MSLN-CAR gene was expressed in CD8-positive cells.

[0062] In addition, 7 days after virus infection, cells were collected, and contaminating plasmid DNA and the like in the virus were digested using Recombinant DNase I (RNAse-free) (manufactured by Takara Bio Inc.). Genomic DNA was then extracted using NucleoSpin (registered trademark) Tissue (manufactured by Machlinergel). Real-time PCR was performed using gene amplification primers specific to the viral vector sequence to quantify the amount of DNA derived from the lentiviral vector. Furthermore, the number of copies of virus integrated into the genome was measured by correcting the DNA amount based on the quantification value of the IFNγ gene. The results are shown in Figure 9. As shown in Figure 9, it was confirmed that the anti-MSLN-CAR gene was integrated into the genome of CD8-positive cells in a manner dependent on the amount of virus.

[0063] Furthermore, among cells infected with the virus solutions LGT2-MSCV and LGT2-EF1a, cells in which 1.97 copies and 0.83 copies, respectively, were introduced into the genome were recovered. Calcein AM (PromoCell) was incorporated into MSLN-positive HeLa cells (ATCC CCL-2) and MSLN-negative K562 cells (JCRB0019), and then co-cultured with the CAR-expressing cells at cell number ratios of 1:1, 3:1, and 10:1 for 4 hours. After co-culture, the culture supernatant was recovered, and the fluorescence intensity was measured to calculate cytotoxicity. The results are shown in Figure 10. As shown in Figure 10, the cytotoxic activity of the CAR-expressing cells against MSLN-positive HeLa cells was confirmed.

[0064] Example 7 Function of siRNA-Expressing Cells Vectors A and D described in WO 2021 / 070956 were prepared. Vector A contains, from 5' to 5', a 5'LTR, a packaging signal sequence, a cPPT sequence, an RRE sequence, an MSCV U3 promoter sequence, SD and SA sequences derived from the human EF1α gene, codon-modified WT1-specific TCRα and β chain gene sequences polycistronically linked by a 2A peptide, and a WPRE sequence. Vector D contains an artificial gene downstream of the WPRE of Vector A that generates four types of siRNA that suppress TCR gene expression. Note that the codons of the codon-modified WT1-specific TCRα and β chain gene sequences have been converted so that their expression is not suppressed by the four types of siRNA. Vectors A and D were designated Vector 1 and Vector 2.

[0065] Next, Vector 3 was prepared by replacing the MSCV-U3 promoter sequence and the SD and SA sequences derived from the human EF1α gene in Vector 1 with a human EF1α promoter sequence. Furthermore, the 5'LTR sequence of Vector 1 was replaced with an LTR sequence (SEQ ID NO: 7) containing a CMV promoter sequence as a foreign promoter sequence to form a hybrid LTR, and 177 bp of the remaining GAG sequence was deleted to form 183 bp (SEQ ID NO: 4). The order of the cPPT sequence and the RRE sequence was swapped, and the WPRE sequence was replaced with WPRE2 (SEQ ID NO: 8) to prepare Vector 5. Vector 4 was prepared by deleting the SD and SA sequences derived from the human EF1α gene in Vector 5. Furthermore, Vector 6 was prepared by replacing the MSCV-U3 promoter sequence of Vector 4 with a human EF1α promoter sequence. The structures of each vector are shown in FIG. 11.

[0066] Using the prepared plasmids, virus solutions were prepared using the same method as in Example 4, and designated virus solutions 1 to 6, respectively. Peripheral blood mononuclear cells (PBMCs) isolated from human peripheral blood were stained with FITC-labeled anti-human CD8 antibody (Becton Dickinson), and CD8-positive cells were isolated using anti-FITC microbeads (MiliTeni Biotec). Virus solutions 1 to 6 were diluted 2-fold, 6-fold, 18-fold, and 54-fold, respectively, and these CD8-positive cells were infected using RetroNectin (Takara Bio). Seven days after virus infection, cells were harvested, and total RNA was extracted using NucleoSpin RNA Plus (Machliner Gel) and treated with DNase I. Using the obtained total RNA as a template, cDNA synthesis was performed using PrimeScript RT reagent Kit (Perfect Real Time) (Takara Bio). Real-time PCR was performed using this cDNA as a template and TB Green Premix Ex Taq II (Takara Bio). The expression levels of the wild-type TCR α chain gene, wild-type TCR β chain gene, codon-modified TCR α chain gene, and codon-modified TCR β chain gene were measured, and the relative values ​​were calculated. Total RNA levels were normalized based on the expression level of the GAPDH gene. Furthermore, the number of virus copies incorporated into the genome was measured using the same method as in Example 6.

[0067] The inhibitory effect of the wild-type TCR gene was evaluated by calculating the relative ratio of gene expression in each experimental group based on the relative values ​​of expression levels of the wild-type TCR α chain gene and wild-type TCR β chain gene in negative control cells not infected with virus. The results are shown in Figure 12. In Figure 12, the vertical axis shows the relative value of gene expression level when the expression level in negative control cells not infected with virus is set to 100. The horizontal axis shows the virus copy number. As shown in the figure, it was confirmed that Vector 1 did not inhibit expression of the wild-type TCR α chain and β chain genes, while Vectors 4, 5, and 6 efficiently inhibited expression.

[0068] In addition, the expression levels of the codon-modified TCR genes were evaluated by calculating the ratio of the relative gene expression levels in each experimental group based on the relative gene expression levels of the codon-modified TCR α chain and the codon-modified TCR β chain in cells infected with a 2-fold diluted virus solution of Vector 1. The results are shown in Figure 13. In Figure 13, the vertical axis indicates the relative gene expression levels when the expression level of the reference group (cells infected with a 2-fold diluted virus solution of Vector 1) is set to 100. The horizontal axis indicates the virus copy number. As shown in Figure 13, it was confirmed that cells transfected with Vector 3 had a low copy number of the introduced virus, while cells transfected with Vectors 5 and 6 expressed equivalent codon-modified human anti-WT1 TCR genes compared to cells transfected with Vectors 2 and 3.

[0069] The present invention provides a nucleic acid construct that efficiently expresses a desired gene, a retroviral vector for introducing the nucleic acid construct into cells, a method for producing a transfected cell using the vector, and a cell into which the vector has been introduced. These nucleic acid constructs, retroviral vectors, methods for producing transfected cells, and transfected cells are extremely useful for protein production, disease treatment by cell therapy, and research and testing for these purposes.

[0070] SEQ ID NO:1: MSCV U3 promoter SEQ ID NO:2: ZsGreen 1 coding sequence SEQ ID NO:3: Vector X gag sequence SEQ ID NO:4: Vector V gag sequence SEQ ID NO:5: Vector W gag sequence SEQ ID NO:6: Vector Y gag sequence SEQ ID NO:7: CMV hybrid LTR SEQ ID NO:8: WPRE2 sequence SEQ ID NO:9: WPRE3 sequence

Claims

1. A nucleic acid construct for producing a retroviral vector, comprising, in order from the 5' end, (a) a 5' LTR (Long Terminal Repeat) sequence derived from a retrovirus containing a foreign promoter sequence, (b) a packaging signal sequence (ψ) derived from a retrovirus, (c) a sequence derived from a nucleic acid encoding a gag protein having a length of 183 to 227 bp, (d) a desired sequence or a multiple cloning site, and (e) a 3' LTR sequence derived from a retrovirus.

2. The nucleic acid construct of claim 1, further comprising a post-transcriptional regulatory sequence (PRE) into which a sequence that causes a frameshift in the nucleic acid sequence encoding the X protein or a stop codon that interrupts translation of the X protein has been inserted.

3. The nucleic acid construct according to claim 2, wherein the PRE is a PRE (WPRE) derived from woodchuck hepatitis virus.

4. The nucleic acid construct according to claim 1, wherein the exogenous promoter sequence is a cytomegalovirus-derived promoter.

5. The nucleic acid construct of claim 1, wherein the desired sequence is a sequence containing an internal promoter sequence.

6. The nucleic acid construct of claim 1, wherein the desired sequence comprises a sequence encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

7. A nucleic acid construct according to claim 6, wherein the desired sequence comprises sequences encoding the TCR alpha and beta chains linked by a 2A peptide.

8. The nucleic acid construct according to claim 1, wherein the LTR sequence is derived from a lentivirus.

9. The nucleic acid construct of claim 1, wherein the 3' LTR sequence is a self-inactivating (SIN) LTR sequence.

10. A retroviral vector comprising a transcript from a nucleic acid construct according to any one of claims 1 to 9.

11. The retroviral vector of claim 10, which comprises a 5'LTR, a packaging signal sequence and a 3'LTR derived from an oncoretrovirus or a lentivirus.

12. A method for producing a retroviral vector, comprising the step of introducing the nucleic acid construct according to any one of claims 1 to 9 into a cell capable of producing retroviral particles.

13. A method for producing a gene-transduced cell, comprising the step of introducing the retroviral vector according to claim 10 or 11 into a cell.

14. The method for producing transgenic cells according to claim 13, wherein the cells are immune cells, cells that can differentiate into immune cells, or cell populations containing them.