WO2023038055A1 - Nucleic acid for retrovirus vector production - Google Patents

Nucleic acid for retrovirus vector production Download PDF

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WO2023038055A1
WO2023038055A1 PCT/JP2022/033543 JP2022033543W WO2023038055A1 WO 2023038055 A1 WO2023038055 A1 WO 2023038055A1 JP 2022033543 W JP2022033543 W JP 2022033543W WO 2023038055 A1 WO2023038055 A1 WO 2023038055A1
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sequence
nucleic acid
cells
acid construct
ltr
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泰典 天石
いづみ 槇
幸子 岡本
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タカラバイオ株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus human T-cell leukaemia-lymphoma virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

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  • the present invention provides a nucleic acid construct for producing a retroviral vector that is used to express a desired gene in mammalian cells, a retroviral vector containing a transcript from the nucleic acid construct, and a gene using the vector. It relates to a method for producing transfected cells and cells containing the nucleic acid construct.
  • Known methods for introducing genes into eukaryotes include methods using viral vectors, techniques for introducing naked DNA by endocytosis, electroporation, and gene guns.
  • Viral vectors are a technology that is widely used in the field of gene therapy from basic to clinical. , is capable of long-term stable expression due to its stable integration function into the host chromosome, and is a promising vector in the field of gene therapy for hereditary diseases, and a technology that is also expected in the field of transgenic animal production. .
  • Retroviruses have gag/pol genes that encode precursor proteins such as viral particle structural proteins, protease, reverse transcriptase, and integrase, and env genes that encode envelope glycoproteins. Both ends of these genes are 5'LTR (Long Terminal Repeat) and 3'LTR involved in transcription of the viral genome, reverse transcription from the viral genome, and integration of the double-stranded DNA synthesized through the reverse transcription into the host DNA. sandwiched between A gene transfer system using retroviral particles consists of one or two packaging constructs expressing the gag/pol and env genes and a retroviral It is divided into transfer vectors that encode RNAs that are incorporated into vector particles.
  • 5'LTR Long Terminal Repeat
  • 3'LTR involved in transcription of the viral genome, reverse transcription from the viral genome, and integration of the double-stranded DNA synthesized through the reverse transcription into the host DNA.
  • sandwiched between A gene transfer system using retroviral particles consists of one or two packaging constructs expressing the gag/pol and env genes
  • the transfer vector has the LTR and the packaging signal sequence for the viral particle, has most of the gag/pol genes and the env gene deleted, and has, for example, the desired sequence inserted.
  • the RNA genome is transcribed by the promoter sequence of the 5'LTR of the transfer vector, and the transcript is packaged into virus particles by the packaging signal sequence.
  • a self-inactivating (SIN) retroviral vector in which the promoter sequence of the 3'LTR that functions as a promoter on the target cell chromosome is deleted. ing.
  • SIN-type vectors transcriptional regulation of the desired gene is performed by an internal promoter located 3' to the packaging signal sequence. Since the SIN vector does not transcribe the sequence on the 5' end side of the internal promoter, the possibility of generating self-replicating viral particles is extremely low and it is safe. So far, vectors with improved safety have been reported (Non-Patent Document 1, Non-Patent Document 2).
  • the purpose of the present invention is to provide a retroviral vector for introducing and expressing foreign genes into cells more safely and efficiently.
  • the present inventors discovered a retroviral vector that can achieve both expression of the introduced gene and safety, and completed the present invention.
  • [1] sequentially from the 5' end, (a) a retrovirus-derived 5' LTR (Long Terminal Repeat) sequence containing an exogenous promoter sequence; (b) a packaging signal sequence ( ⁇ ) from a retrovirus, (c) a sequence derived from a nucleic acid encoding a gag protein between 183 and 227 bp in length; (d) a desired sequence or multiple cloning site; (e) a 3'LTR sequence from a retrovirus; A nucleic acid construct for producing a retroviral vector, comprising each sequence of [2] The nucleic acid construct of [1], further comprising a post-transcriptional regulatory sequence (PRE) inserted with a sequence that causes a frameshift in the nucleic acid sequence encoding the X protein or a termination codon that interrupts the translation of the X protein.
  • PRE post-transcriptional regulatory sequence
  • WPRE woodchuck hepatitis virus-derived PRE
  • the nucleic acid construct of [1], wherein the desired sequence comprises a sequence encoding a T-cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • TCR T-cell receptor
  • CAR chimeric antigen receptor
  • a retroviral vector comprising a transcript from the nucleic acid construct of any one of [1] to [9].
  • the retroviral vector of [10] comprising a 5'LTR, a packaging signal sequence and a 3'LTR derived from an oncoretrovirus or lentivirus.
  • a method for producing a retroviral vector comprising the step of introducing the nucleic acid construct of any one of [1] to [9] into a cell capable of producing retroviral particles.
  • a method for producing a gene-introduced cell comprising the step of introducing the retroviral vector of [10] or [11] into a cell.
  • a highly safe nucleic acid construct for producing a retrovirus a retroviral vector comprising a transcription product from the nucleic acid construct, and the vector are used to efficiently express a desired gene.
  • a method for producing a transgenic cell and a cell containing the nucleic acid construct are provided.
  • FIG. 2 is a diagram showing the structure of a nucleic acid construct prepared in Examples.
  • FIG. 2 shows relative fluorescence intensity values of ZsGreen1 protein expressed by cells infected with retroviral vectors produced using respective nucleic acid constructs.
  • FIG. 3 shows the relative viral titers of retroviral solutions produced using each nucleic acid construct.
  • FIG. 2 is a diagram showing the structure of a nucleic acid construct prepared in Examples.
  • FIG. 4 shows the virus titers of retroviral solutions produced using each nucleic acid construct.
  • FIG. 2 is a diagram showing the structure of a nucleic acid construct prepared in Examples.
  • FIG. 4 shows the virus titers of retroviral solutions produced using each nucleic acid construct.
  • FIG. 1 shows the structure of a nucleic acid construct prepared in Examples.
  • FIG. 4 shows the virus titers of retroviral solutions produced using each nucleic acid construct.
  • FIG. 2 shows the proportion of cells positive for CAR protein expression among cells infected with retroviral vectors produced using each nucleic acid construct.
  • FIG. 2 shows the number of viral copies integrated into the genome of cells infected with retroviral vectors produced using each nucleic acid construct.
  • FIG. 3 shows cytotoxic activity of CAR-expressing cells infected with retroviral vectors produced using each nucleic acid construct.
  • FIG. 2 is a diagram showing the structure of a nucleic acid construct prepared in Examples.
  • FIG. 2 shows RNA expression levels of wild-type TCR proteins expressed by cells infected with retroviral vectors produced using respective nucleic acid constructs.
  • FIG. 2 shows RNA expression levels of codon-converted TCR proteins expressed by cells infected with retroviral vectors produced using respective nucleic acid constructs.
  • nucleic acid construct means a nucleic acid containing a sequence constructed to contain one or more functional units not found in nature.
  • Nucleic acids can be DNA and/or RNA and can also include modified nucleic acids. Forms include circular, linear, double-stranded, single-stranded, extrachromosomal DNA molecules (plasmids), cosmids, and the like.
  • Nucleic acid constructs also include a nucleic acid sequence encoding a gene, optionally including a control sequence (e.g., promoter) operably linked (i.e., capable of controlling transcription or translation). can contain.
  • the nucleic acid construct may optionally contain other regulatory elements, functional sequences, linkers, and the like.
  • LTR Long Terminal Repeat
  • the LTR is composed of U3, R, and U5 regions 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 4-20 base pairs long.
  • U3 contains transcriptional enhancer and promoter sequences.
  • the “packaging signal sequence” is also referred to as “psi sequence” or “ ⁇ sequence” and is required for encapsidation of retroviral RNA strands and packaging into viral particles in the formation of viral particles. , refers to non-coding cis-acting sequences. For example, the region 3' of the major splice donor (SD) site to the gag initiation codon, or the region 3' of the SD site that includes part of the gag gene sequence.
  • SD major splice donor
  • sequence of interest refers to an artificial (by artificial manipulation) insertion into a cell (e.g., the nuclear genome or cytoplasm of a cell), either transiently or permanently. means an exogenous sequence that is desired to be Such sequences include gene sequences that are wholly or partially heterologous to the cell into which they are introduced, and also include gene sequences with any mutations. It may also be the same gene sequence as the endogenous gene that the cell naturally has.
  • naturally means being in a natural state without any artificial manipulation.
  • a "multicloning site” is a cluster sequence consisting of multiple restriction enzyme sites for cloning. There are no particular restrictions on the nucleotide sequence that constitutes the multicloning site and the type and number of restriction enzyme sites included.
  • posttranscriptional regulatory element refers to promoting polyadenylation of mRNA transcribed from a gene in a cell, promoting nuclear export of mRNA, or activating translation of mRNA. indicates a sequence that contributes to By inserting into the untranslated region of the desired gene contained in the nucleic acid construct of the present invention, expression of the desired gene at the protein level is enhanced.
  • wild-type means 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 made artificially.
  • T cells are also called T lymphocytes, and mean cells derived from the thymus among lymphocytes involved in immune response.
  • T cells include helper T cells, suppressor T cells, regulatory T cells, CTL, naive T cells, memory T cells, ⁇ T cells expressing ⁇ and ⁇ chain TCRs, and ⁇ and ⁇ chain TCRs. It includes ⁇ T cells that "Cells that can differentiate into T cells” are not particularly limited as long as they are cells that differentiate into T cells in vivo or by artificial stimulation. Included are progenitor cells, T-cell progenitor cells, and the like.
  • the "cell population containing T cells or cells capable of differentiating into T cells” includes blood (peripheral blood, umbilical cord blood, etc.), bone marrow fluid, and peripheral blood collected, isolated, purified, and induced therefrom.
  • Cell populations including nuclear cells (PBMC), hematopoietic cells, hematopoietic stem cells, cord blood mononuclear cells and the like are exemplified.
  • PBMC nuclear cells
  • hematopoietic cells hematopoietic stem cells
  • cord blood mononuclear cells and the like are exemplified.
  • Various cell populations derived from blood lineage cells containing T cells can also be used in the present invention. These cells may be activated in vivo or ex vivo by cytokines such as anti-CD3 antibodies and IL-2. These cells can be either collected from a living body or obtained through in vitro culture, for example, a T cell population obtained from a living body as it is or cryopreserved
  • Nucleic acid construct of the present invention (a) a retrovirus-derived 5' LTR (Long Terminal Repeat) sequence containing an exogenous promoter sequence; (b) a packaging signal sequence ( ⁇ ) from a retrovirus, (c) a sequence derived from a nucleic acid encoding a gag protein between 183 and 227 bp in length; (d) a desired sequence or multiple cloning site; (e) a 3'LTR sequence from a retrovirus; is a nucleic acid construct for producing a retroviral vector, comprising each sequence of The nucleic acid constructs of the invention can be used to produce retroviral vectors.
  • a retrovirus-derived 5' LTR Long Terminal Repeat
  • the retroviral vector of the present invention containing a transcript from the nucleic acid construct can be produced.
  • the nucleic acid constructs of the present invention are capable of producing high viral titer retroviral vectors. This retroviral vector allows the desired sequences to be introduced into the cell. Cells into which a desired sequence has been introduced by the retroviral vector of the present invention have a high expression efficiency of the desired sequence.
  • a retrovirus is an enveloped virus having a genome of positive-strand single-stranded RNA, and the viral genome includes, from its 5' end, a 5'LTR sequence, an SD sequence, a packaging signal sequence, a gag gene, a pol. Gene, SA sequence, env gene, 3'LTR sequence are present. In the case of lentivirus, which will be described later, a plurality of accessory genes are included in addition to these elements. Among them, in the retroviral vector-based gene transfer system, the 5'LTR sequence, the packaging signal sequence, and the 3'LTR sequence are essential for the retroviral vector, and the nucleic acid of the present invention comprises all of these. Other gene products such as gag, pol, env, etc. can be supplied from packaging cells carrying these genes. Generally, the desired sequence is placed 3' to the packaging signal sequence of the retroviral vector, or 3' to both the packaging signal sequence and the SA sequence if the SA sequence is present.
  • a nucleic acid construct of the present invention comprises a sequence derived from a nucleic acid encoding a gag protein with a length of 183-227 bp.
  • the retroviral packaging signal sequence partially includes the sequence of the gag gene and cannot be completely removed, a sequence of this length is useful for efficient virus production and expression of the desired sequence. .
  • the present invention can provide a method for producing a highly safe viral vector.
  • a sequence derived from a nucleic acid encoding a gag protein is suitably 183 bp or 227 bp in length and is the sequence set forth in SEQ ID NO: 4 or 3, or one or several, such as 1-9 are substituted, deleted, inserted or added.
  • the (a) 5'LTR sequence containing the exogenous promoter sequence, (e) the 3'LTR sequence and (b) the packaging signal sequence contained in the nucleic acid construct of the present invention are retrovirus-derived sequences and comprise these sequences. Any sequence capable of producing a retrovirus containing the RNA as its genome can be used. Retroviruses include subclasses of oncoretroviruses and lentiviruses, and sequences from either class of virus can be used in the present invention. These sequences may be sequences derived from the same virus, but sequences derived from different viruses may be combined to the extent that viral particles can be formed and integration into the genome of the transfected cell can be achieved by combination with an appropriate packaging cell. may be used.
  • LTR sequences and packaging signal sequences used in the present invention include, for example, Moloney murine leukemia virus (MMLV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), myeloproliferative sarcoma, which belongs to the oncoretroviruses. Sequences from viruses (MPSV), splenic focal-focal virus (SFFV) can be used.
  • Oncoretrovirus-derived viral vectors are capable of highly efficient gene transfer, but cells must be actively undergoing cell division at the time of vector transfer.
  • LTR sequence and packaging signal sequence used in the present invention include, for example, human immunodeficiency virus (HIV-1, HIV-2) belonging to lentiviruses, simian immunodeficiency virus (SIV), feline immunodeficiency virus ( FIV), Equine Infectious Anemia Virus (EIAV), Captive Arthritis Encephalitis Virus (CAEV) can be used.
  • Lentiviral vectors can introduce genes into the genome in the nucleus regardless of mitosis of the cells into which they are introduced. Lentiviral vectors have also been described in numerous publications [eg, J. Virology, 72:8463-8471 (1998)].
  • Other groups of retroviruses, such as spumaviruses eg, foamy viruses
  • 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 RNA polymerase II of the host cell.
  • the 5'LTR sequences used in the present invention contain enhancers/promoters that are foreign to the virus from which the LTR is derived.
  • the U3 region of the 5'LTR is replaced with an exogenous enhancer/promoter.
  • an LTR sequence in which the U3 region of the 3'LTR sequence is replaced with an enhancer/promoter derived from a virus other than the virus from which the LTR sequence is derived can also be used in the present invention.
  • the exogenous enhancer/promoter to replace can be of viral or mammalian origin and can be constitutive, inducible or tissue-specific.
  • virus-derived enhancers/promoters such as human cytomegalovirus (HCMV) immediate early, Moloney murine sarcoma virus (MMSV), mouse stem cell virus (MSCV), Rous sarcoma virus (RSV), spleen focus-forming virus (SFFV), etc.
  • Enhancer/promoter refers to sequences comprising enhancer and/or promoter regions.
  • enhancer regions and promoter regions are sometimes collectively referred to as promoters.
  • the promoter region is sometimes called a core promoter. Either enhancer regions and/or promoter regions can be used in the present invention.
  • the 5' LTR sequence containing the foreign promoter sequence is the sequence set forth in SEQ ID NO: 7, or one or several, for example, 1 to 9, base substitutions, deletions, or substitutions in this sequence. Including inserted or added base sequences.
  • a sequence in which enhancer/promoter activity is deleted by introducing mutations into the U3 region can be used.
  • a retroviral vector in which the U3 region of the 3'LTR sequence is mutated is called a self-inactivating (SIN) vector. Mutations are introduced into the 3'LTR sequence by base substitution or deletion.
  • a promoter sequence separate from the LTR is arranged.
  • Such a promoter sequence existing between the 5'LTR and the 3'LTR is sometimes referred to as an internal promoter sequence.
  • the internal promoter sequence promoter sequences derived from viruses or mammalian genes can be used. If a viral promoter sequence is used, it can be 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.
  • a promoter sequence derived from the U3 region of the LTR of a retrovirus such as a promoter sequence derived from the U3 region of the LTR of a mouse stem cell virus (MSCV), or the like can be used.
  • the sequence exemplified as the exogenous promoter that replaces the U3 region of the 5'LTR sequence in the preceding paragraph can be used.
  • a virus-derived promoter sequence in addition to the above, sequences such as SV40 promoter and CMV promoter can be used.
  • sequences such as phosphoglycerate kinase (PGK) promoter, polypeptide chain elongation factor (EF1- ⁇ ) promoter, ⁇ -actin promoter, and CAG promoter, which are promoters that function in mammalian cells, can also be used.
  • the internal promoter may be placed upstream of (d) (that is, on the 5' side), and should be placed between (a) and (d), preferably between (b) and (d). can be done.
  • the nucleic acid construct of the present invention may contain an SD sequence and/or an SA sequence. These sequences can be SD and/or SA sequences exogenous to the LTR, or SD and/or SA sequences exogenous to the internal promoter sequence. Moreover, the SD sequence and the SA sequence may be sequences of different origins. For example, simian virus (SV) 40 16S RNA, HCMV immediate early RNA, human hEF1 ⁇ gene-derived SD and SA sequences can be used [Proceedings of the National Academy of Sciences, Vol. 95, No. 1, 219-223. (1998)]. Moreover, the SD sequence or SA sequence, in which the consensus sequence is mutated to enhance or suppress the splicing activity, can also be used in the nucleic acid construct of the present invention.
  • SV simian virus
  • 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.
  • sequences include, for example, sequences encoding proteins, and sequences encoding RNAs that function in cells, such as tRNAs and miRNAs.
  • a nucleic acid construct is prepared in which a sequence (d) in which a plurality of restriction enzyme recognition sequences for ligating desired sequences are arranged (multicloning site) is arranged, and then the desired sequence is obtained using the multicloning site. may be inserted.
  • Such nucleic acid constructs having multiple cloning sites in place of desired sequences are also included in the nucleic acid constructs of the present invention.
  • the desired sequence may be for the purpose of disease prevention or treatment.
  • a sequence useful for suppressing the transcription or expression of a gene product that is harmful in vivo for example, a sequence encoding siRNA), or a protein that is used to replenish a protein that has been deleted or has lost its function in vivo.
  • Sequences, sequences capable of modifying or enhancing functions of cells, and the like are exemplified.
  • the present invention provides gene therapy in which cells into which a foreign sequence has been introduced by the nucleic acid construct of the present invention are introduced into a living body.
  • sequences encoding the IL-2 receptor ⁇ chain (X-linked severe combined immunodeficiency), sequences encoding ⁇ -globin ( ⁇ -thalassemia), sequences encoding adenosine deaminase (ADA) (ADA deficiency), blood Gene therapy using sequences encoding clotting factors (hemophilia), sequences encoding receptors that recognize antigens (cancer and viral infections) are exemplified.
  • 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.
  • the oligomer protein may be a cell surface protein (membrane protein), and a sequence encoding an antigen recognition receptor exemplified in the Examples, such as a T cell receptor (T cell receptor: TCR) is desirable. is suitable as an array of
  • the desired sequence contained in the nucleic acid construct of the present invention is a sequence encoding a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • a typical CAR structure consists of a single chain antibody (single chain variable fragment: scFv) that recognizes surface antigens of target cells to be eliminated from the body, for example, tumor cells, a transmembrane domain, and an intracellular domain that activates T cells. Configured.
  • the intracellular domain the intracellular domain of the TCR complex CD3zeta is preferably used.
  • a CAR with such a configuration is called a first generation CAR.
  • the genes for single-chain antibody portions can be isolated, for example, from hybridomas that produce monoclonal antibodies that recognize the target antigen.
  • CAR-expressing T cells directly recognize the surface antigens of target cells, irrespective of the expression of major histocompatibility class I antigens on tumor cells, and simultaneously activate T cells to efficiently kill target cells. It is possible to
  • first-generation CARs With the aim of enhancing the ability of first-generation CARs to activate T cells, second-generation CARs linked to the intracellular domains of T-cell co-stimulatory molecules have been developed.
  • co-stimulatory molecules for T cells CD28, the intracellular domain of CD137 (4-1BB) or CD134 (OX40) of the tumor necrosis factor (TNF) receptor superfamily is preferably used.
  • third-generation CARs in which the intracellular domains of these co-stimulatory molecules are linked in tandem have also been developed, and many CAR molecules targeting various tumor antigens have been reported.
  • the nucleic acid constructs of the invention can contain sequences encoding any CAR as desired sequences.
  • the nucleic acid constructs of the present invention may contain post-transcriptional regulatory sequences (PREs), which are useful for enhancing expression of desired sequences.
  • 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 are the post-transcriptional processing elements of herpes simplex virus, the post-transcriptional regulatory sequences of hepatitis B virus (HPRE) and woodchuck hepatitis virus (WPRE).
  • the PRE sequence contains the X protein coding region, which has been pointed out to be carcinogenic.
  • Nucleic acid constructs of the present invention can also use mutant sequences in which the expression of the X protein from the PRE sequence is suppressed.
  • a PRE sequence with an inserted stop codon that causes a frameshift in the nucleic acid sequence encoding the X protein or a stop codon that interrupts translation of the X protein can be used.
  • a PRE sequence containing one or two inserted bases that causes a frameshift between positions 6 and 7 with A of the initiation codon ATG of the X protein as position 1 can be used.
  • a PRE sequence in which positions 7 to 9 are replaced with stop codons eg, TAA
  • WPRE is preferred for the present invention. WPRE can be referred to US Pat.
  • WPRE2 SEQ ID NO: 8
  • WPRE3 SEQ ID NO: 9
  • a nucleotide sequence in which one or several, for example 1 to 9, bases are substituted, deleted, inserted or added to this sequence can be used.
  • One aspect of the present invention is a nucleic acid construct in which a PRE sequence is positioned between (d) a desired sequence or multiple cloning site and (e) a 3'LTR sequence derived from a retrovirus.
  • the nucleic acid constructs of the present invention may contain Rev response element (RRE) sequences and/or central polypurine tract (cPPT) sequences.
  • RRE Rev response element
  • cPPT central polypurine tract
  • RRE include, but are not limited to, RREs such as those located at positions 7622-8459 of the HIV NL4-3 genome (GenBank Accession Number: AF003887), RREs derived from other strains of HIV or other retroviruses. do not have.
  • cPPT is a sequence of about 15 bases that exists almost in the center of the lentiviral genome, and serves as a primer binding site for synthesizing plus-strand DNA in the process of synthesizing double-stranded DNA from lentiviral genomic RNA. .
  • the lentiviral RNA genome When the lentiviral RNA genome is reverse transcribed, it works with the central termination sequence (CTS) to form a triple-stranded structure called a DNA flap.
  • CTS central termination sequence
  • the arrangement of both sequences is not particularly limited, and may be arranged in the order of cPPT and RRE or the order of RRE and cPPT from the 5' end.
  • the present invention includes a method for producing a retroviral vector (particle), which includes the step of introducing the nucleic acid construct of the present invention into a cell capable of producing retroviral particles.
  • a nucleic acid construct can be introduced into cells using a suitable vector, such as a plasmid vector, a viral vector other than a retrovirus, or a transposon vector, so that it can stably exert its effect in the cell. Additionally, it can be integrated onto the chromosomal DNA of the cell using genome editing techniques. Alternatively, the nucleic acid construct can be directly introduced into cells.
  • Methods using carriers such as liposomes and ligand-polylysine, calcium phosphate methods, electroporation methods, particle gun methods, and the like can be used to introduce nucleic acid constructs into cells directly or with a plasmid vector.
  • the viral vector is not particularly limited, and is usually a known viral vector used in gene transfer methods, such as adenovirus vector, adeno-associated virus vector, simian virus vector, vaccinia virus vector, measles virus vector or Sendai virus vector. etc. are used.
  • cells capable of producing retroviral particles for example, known packaging cells can be used.
  • An appropriate packaging cell is selected based on the LTR sequence and packaging signal sequence possessed by the nucleic acid construct, the nucleic acid construct is introduced into the retrovirus-producing cell, and a retroviral vector (particle) is prepared. can do.
  • cells with high transfection efficiency (293 cells, 293T cells, etc.) may be encoded with the nucleic acid construct of the present invention and components (gag gene, pol gene, env gene and other accessory genes) necessary for retroviral particle production.
  • the retroviral particles can be produced by simultaneously or optionally introducing the nucleic acids that are capable of producing retroviral particles and culturing the cells in a suitable medium as retroviral producer cells.
  • the retroviral particles produced contain transcripts from the nucleic acid constructs of the invention.
  • This retroviral particle is included in the present invention as a retroviral vector for introducing desired sequences into cells.
  • packaging cells include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), and the like.
  • Packaging cells, other cells, kits containing retrovirus-producing plasmids (referred to as packaging plasmids), and the like are widely commercially available from various companies, and these can be used in the method of the present invention.
  • packaging cell expressing an envelope protein derived from a virus heterologous to that from which the genome of the retroviral vector is derived, or a packaging plasmid containing a sequence encoding the envelope protein
  • Pseudotyped retroviruses can be produced.
  • packaging plasmids encoding envelopes derived from MMLV, gibbon leukemia virus (GaLV), vesicular stomatitis virus (VSV), feline endogenous virus or proteins capable of functioning as envelopes can be used.
  • retroviral vectors having sugar chain-modified proteins on their surfaces can be produced using packaging cells into which enzyme genes involved in sugar chain synthesis have been introduced.
  • the cell culture After culturing the retrovirus-producing cells created by the above operation, the cell culture is centrifuged to collect the supernatant, and contaminants can be removed by appropriate filtration to obtain retrovirus particles.
  • Gene transfer can be performed by directly contacting the crude retroviral particles with cells, but retroviral particles of higher purity may be prepared through known purification procedures and subjected to gene transfer procedures.
  • the present invention provides a composition comprising the retroviral vector of the present invention as an active ingredient together with a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable excipients include, for example, phosphate-buffered saline (eg, 0.01 M phosphate, 0.138 M NaCl, 0.0027 M KCl, pH 7.0). 4), aqueous solutions containing mineral salts such as hydrochlorides, hydrobromides, phosphates, sulfates, physiological saline, solutions such as glycol or ethanol, and acetates, propionates, malonates, Contains salts of organic acids such as benzoate.
  • Adjuvants such as wetting or emulsifying agents, and pH buffering agents can also be used.
  • pharmaceutically acceptable excipients those described in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991) (incorporated herein by reference) are used as appropriate.
  • the composition can be in any known form suitable for parenteral administration, eg injection or infusion. Additionally, formulation aids such as suspending, preserving, stabilizing and/or dispersing agents, and preservatives can be used to extend shelf life during storage.
  • the composition may be in dry form for reconstitution with a suitable sterile liquid before use.
  • a method for producing a gene-introduced cell of the present invention comprises transferring a retroviral vector containing the nucleic acid construct of the present invention described in (1) above or a transcript from the nucleic acid construct into a cell. characterized by comprising a step of introducing into 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 transfected cell of the present invention. In one aspect of the invention, the process is performed outside the body.
  • the method 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.
  • Cells used in the method of the present invention are not particularly limited, and any cells can be used.
  • cells collected, isolated, purified, and induced from blood peripheral blood, umbilical cord blood, etc.
  • bodily fluids such as bone marrow, tissues or organs can be used.
  • PBMC Peripheral blood mononuclear cells
  • immune cells T cells, dendritic cells, B cells, hematopoietic stem cells, macrophages, monocytes, NK cells or blood cells (neutrophils, basophils)
  • cord blood alone Nucleocytes, fibroblasts, preadipocytes, hepatocytes, skin keratinocytes, mesenchymal stem cells, adipose stem cells, various cancer cell lines or neural stem cells can be used.
  • immune cells immune cell progenitor cells (hematopoietic stem cells, lymphocyte progenitor cells, etc.), or cell populations containing these cells.
  • T cells which are representative of immune system cells, include ⁇ T cells, ⁇ T cells, CD8 positive T cells, CD4 positive T cells, regulatory T cells, cytotoxic T cells, or tumor-infiltrating lymphocytes.
  • Cell populations containing T cells and T cell progenitor cells include PBMCs.
  • NK cells, NKT cells, and progenitor cells thereof can also be targeted by the methods of the present invention. Examples of the above-mentioned cells include, but are not limited to, those collected from living organisms, those obtained by expanding and culturing them, those established as cell lines, those differentiated from pluripotent stem cells, and the like. When it is desired to transplant the produced gene-introduced cells or cells differentiated from the cells into a living body, it is preferable to produce the gene-introduced cells from cells harvested from the living body itself or from the same kind of living body.
  • fibronectin or fibronectin fragments are examples of viral vectors.
  • fibronectin fragment having a heparin-binding site such as a fragment commercially available as RetroNectin (registered trademark, CH-296, manufactured by Takara Bio Inc.) can be used.
  • RetroNectin registered trademark, CH-296, manufactured by Takara Bio Inc.
  • retroviral gene transfer aids may be used.
  • the functional substance may be used by a method suitable for each substance. flasks, bags, etc.) or immobilized on carriers (microbeads, etc.).
  • the cell of the present invention is a gene-introduced cell produced by the production method of (2) above.
  • the cells of the invention express the gene product encoded by the exogenous desired sequence.
  • the transgenic cells of the present invention have acquired new properties and/or functions resulting from said gene product.
  • the cells of the present invention can be used as therapeutic agents for diseases.
  • the therapeutic agent contains, as an active ingredient, the cells of the present invention capable of expressing gene products useful for treating diseases, and may further contain suitable excipients.
  • the excipient is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include stabilizers, buffers, tonicity agents and the like.
  • Diseases to which the cells of the present invention are administered are not particularly limited as long as they are diseases that exhibit sensitivity to the cells, but examples include cancer [blood cancer (leukemia), solid tumors, etc.] and inflammatory diseases.
  • the cells of the present invention can also be used for bone marrow transplantation, prevention of post-irradiation infections, transfusion of donor lymphocytes for the purpose of remission of recurrent leukemia, etc.
  • a therapeutic agent comprising the cells of the present invention as an active ingredient is: , but not limited to parenteral administration, such as by injection or infusion, intradermally, intramuscularly, subcutaneously, intraperitoneally, intranasally, intraarterially, intravenously, intratumorally, or intraafferent lymphatics. be able to.
  • Example 1 Preparation of Gag Residual Sequence-Reduced Viral Vector Plasmid First, using pMSCVneo (manufactured by Clontech) as a template, a DNA fragment of the MSCV U3 promoter sequence shown in SEQ ID NO: 1 was amplified by PCR. Subsequently, using pLVSIN-IRES-ZsGreen1Vector as a template, a DNA fragment having the ZsGreen1 sequence shown in SEQ ID NO: 2 was amplified by PCR.
  • Example 2 Preparation of Retrovirus Solution
  • E. coli JM109 was transformed with pLVSIN-MSCV-ZsGreen1 prepared in Example 1 and vectors X, V, W, Y and Z, respectively.
  • Plasmid DNAs carried by these transformants were purified using NucleoSpin (registered trademark) Plasmid Midi (manufactured by Mach Reiner Gel) and subjected to the following operations as DNAs for transfection.
  • Each prepared plasmid and packaging plasmid (in which HIV-1-derived Gag, Pol, Tat, and Rev lentiviral proteins and VSV-G envelope protein are transiently expressed) were transferred to 293T cells (ATCC CRL- 11268), and the resulting cells were cultured to prepare supernatants containing lentiviruses having VSV-G envelopes. The supernatant was filtered through a 0.45 ⁇ m filter (Milex HV, manufactured by Millipore) to obtain virus solution LVSIN-MSCV-ZsGreen1, solution X, solution Y, solution V, solution W, and solution Z, respectively.
  • Milex HV manufactured by Millipore
  • Example 3 Infection with Gag sequence-reduced lentiviral vector
  • the virus solution prepared in Example 2 was appropriately diluted, and a human T-lymphocytic leukemia-derived cell line SupT1 cell (ATCC CRL-1942) was infected once.
  • a flow cytometer the percentage of ZsGreen1-positive cells in cells 3 and 4 days after virus infection and the average fluorescence intensity in the positive cells were measured, and the virus titer was calculated according to the following formula.
  • Virus titer number of infected cells x (% positive rate/100) x virus dilution rate/liquid volume at time of infection (mL)
  • Figure 2 shows a comparison of the average fluorescence intensity when the vector LVSIN-MSCV-ZsGreen1 is 100%
  • Figure 3 shows a comparison of the virus titers. All are calculating the average value of the value tested 3 times.
  • vectors X, V, W, and Y exhibited mean fluorescence intensities comparable to vector LVSIN-MSCV-ZsGreen1.
  • vectors W and Y showed a significant decrease in virus titer, while vectors X and V did not show a significant decrease.
  • Example 4 Hybrid LTR and WPRE2 pLVSIN-EF1 ⁇ -ZsGreen1 was prepared by replacing the MSCV-U3 promoter sequence of pLVSIN-MSCV-ZsGreen1 prepared in Example 1 with the human EF1 ⁇ 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 an exogenous promoter sequence to obtain a hybrid LTR, and the remaining GAG sequence was deleted by 177 bp to 183 bp. (SEQ ID NO: 4).
  • the WPRE sequence is WPRE2 in which an insertion base that causes a frameshift between positions 6 and 7 with A of the start codon ATG of the X protein as 1 is inserted in order to suppress the expression of the X protein contained in the sequence.
  • SEQ ID NO: 8 The structure of each vector is shown in FIG.
  • Escherichia coli HST08 was transformed with each of the prepared plasmid DNAs. Plasmid DNA retained by these transformants was purified using NucleoSpin (registered trademark) Plasmid Midi (manufactured by Mach Reiner Gel), and subjected to the following operations as DNA for transfection.
  • NucleoSpin registered trademark
  • Plasmid Midi manufactured by Mach Reiner Gel
  • Each of the prepared plasmid DNAs and the packaging plasmid used in Example 2 were transfected into 293T cells to obtain four supernatants containing VSVG enveloped lentiviruses.
  • pLGT2-MSCV-ZsGreen1 and pLGT2-EF1 ⁇ -ZsGreen1 a packaging plasmid containing no plasmid expressing the lentiviral TAT protein was used. The supernatant was filtered through a 0.45 ⁇ m filter (Milex HV, manufactured by Millipore) to prepare virus solutions, LVSIN-MSCV, LVSIN
  • FIG. 5 shows the results of calculating the virus titer using the measured values at which the ZsGreen1 positive rate was 1.0-20.0%. As shown in FIG. 5, equivalent viral titers were obtained for all lentiviral vectors.
  • the A of the start codon ATG of the X protein is the 1st position and the 7th to 9th positions are the stop codon (TAA A plasmid DNA was prepared containing WPRE3 (SEQ ID NO: 9) substituted with ).
  • a virus solution was prepared in the same manner as described above and infected with J45.01 cells to confirm that equivalent virus titers were obtained.
  • a sequence encoding the fluorescent protein AcGFP was ligated to the 3′ ends of the wild-type WPRE sequence, WPRE2 and WPRE3, respectively, and the expression of the fusion protein of the X protein and AcGFP was confirmed. bottom. Fluorescence intensity measurements showed that X protein was not expressed from WPRE2 and WEPRE3, confirming the high safety of WPRE2 and WEPRE3.
  • Example 5 Expression of CAR gene
  • the ZsGreen1 sequence of each vector plasmid DNA prepared in Example 4 is replaced with a sequence (MSLN-CAR) encoding a chimeric antigen receptor (CAR) that specifically recognizes mesothelin (MSLN).
  • a plasmid DNA was prepared.
  • pLVSIN-MSCV-MSLN-CAR, pLVSIN-EF1 ⁇ -MSLN-CAR, pLGT2-MSCV-MSLN-CAR and pLGT2-EF1 ⁇ -MSLN-CAR respectively.
  • the structure of each vector is shown in FIG.
  • FIG. 7 shows the results of measuring the virus titer in the same manner as in Example 4.
  • the lentiviral vector prepared with the plasmid DNA containing the hybrid LTR showed equivalent or higher viral titer than the conventional lentiviral vector.
  • Example 6 Functions of CAR Gene-Introduced Cells Using the hybrid LTR-containing plasmid DNA prepared in Example 5, virus solutions LGT2-MSCV and LGT2-EF1 ⁇ containing MSLN-CAR sequences were isolated from human peripheral blood. The obtained peripheral blood mononuclear cells (PBMC) were infected with RetroNectin (manufactured by Takara Bio Inc.) at various dilutions.
  • PBMC peripheral blood mononuclear cells
  • Vector A consists of, in order from 5′, 5′ LTR, packaging signal sequence, cPPT sequence, RRE sequence, MSCV U3 promoter sequence, human EF1 ⁇ gene-derived SD and SA sequences, codon conversion linked polycistronic by 2A peptide. Includes type WT1-specific TCR ⁇ and ⁇ chain gene sequences, WPRE sequences.
  • Vector D contains, downstream of WPRE of vector A, artificial genes that generate four types of siRNA that suppress TCR gene expression.
  • the codon-converted WT1-specific TCR ⁇ -chain and ⁇ -chain gene sequences are codon-converted so that their expression is not suppressed by the four types of siRNA.
  • Said vector A and vector D were referred to as vector 1 and vector 2, respectively.
  • Vector 3 was prepared by replacing the MSCV-U3 promoter sequence of Vector 1 and the SD and SA sequences derived from the human EF1 ⁇ gene with the human EF1 ⁇ 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 obtain a hybrid LTR, and 177 bp of the remaining GAG sequence was deleted to 183 bp (SEQ ID NO: 4), the order of the cPPT sequence and the RRE sequence was reversed, and vector 5 was prepared by replacing the WPRE sequence with WPRE2 (SEQ ID NO: 8).
  • LTR sequence SEQ ID NO: 7
  • Vector 4 was constructed by deleting the SD and SA sequences derived from the human EF1 ⁇ gene of Vector 5.
  • vector 6 was constructed by replacing the MSCV-U3 promoter sequence of vector 4 with the human EF1 ⁇ promoter sequence. The structure of each vector is shown in FIG.
  • each virus solution was prepared in the same manner as in Example 4 to obtain virus solutions 1 to 6, respectively.
  • Peripheral blood mononuclear cells (PBMC) isolated from human peripheral blood were stained with FITC-labeled anti-Human CD8 antibody (manufactured by Becton Dickinson), and CD8 was detected with anti-FITC microbeads (manufactured by Militeni Biotech). Positive cells were isolated.
  • Virus solutions 1 to 6 were diluted 2-fold, 6-fold, 18-fold and 54-fold, respectively, and retroNectin (manufactured by Takara Bio Inc.) was used to infect these CD8-positive cells.
  • cDNA synthesis was performed using PrimeScript RT reagent Kit (Perfect Real Time) (manufactured by Takara Bio Inc.).
  • real-time PCR was performed using TB Green Premix Ex Taq II (manufactured by Takara Bio), wild-type TCR ⁇ chain gene, wild-type TCR ⁇ chain gene, codon-converted TCR ⁇ chain gene, codon-converted TCR ⁇ chain.
  • the expression level of the gene was measured and the relative value was calculated.
  • the total RNA amount was corrected based on the expression level of the GAPDH gene.
  • the virus copy number integrated into the genome was measured.
  • the ratio of the relative values of gene expression in each experimental group was calculated to obtain the wild-type TCR gene. was evaluated for its inhibitory effect.
  • FIG. 12 the vertical axis indicates the relative value of the expression level of the gene when the expression level of the negative control cells not infected with the virus is set to 100.
  • the horizontal axis indicates the virus copy number.
  • vector 1 did not suppress the expression of wild-type TCR ⁇ chain and ⁇ chain genes, and vectors 4, 5 and 6 efficiently suppressed the expression.
  • relative values of gene expression of codon-converted TCR ⁇ chain and codon-converted TCR ⁇ chain in cells infected with 2-fold diluted vector 1 virus solution are used as reference, and ratio of relative values of gene expression in each experimental group.
  • the expression level of the codon-converted TCR gene was evaluated by calculating .
  • the results are shown in FIG. In FIG. 13 , the vertical axis indicates the relative value of the expression level of the gene when the expression level of the reference (cells infected with the 2-fold diluted vector 1 virus solution) is set to 100.
  • the horizontal axis indicates the virus copy number. As shown in FIG.
  • the cells into which vector 3 was introduced had a lower copy number of the introduced virus, and the cells into which vectors 5 and 6 were introduced had equivalent codon-conversion types than the cells into which vectors 2 and 3 were introduced. It was confirmed that the human anti-WT1 TCR gene was expressed.
  • the present invention provides a nucleic acid construct that efficiently expresses a desired gene, a retroviral vector for introducing the nucleic acid construct into a cell, a method for producing a gene-introduced cell using the vector, and a cell into which the vector has been introduced. be done.
  • These nucleic acid constructs, retroviral vectors, methods for producing gene-introduced cells, and gene-introduced cells are extremely useful for protein production, treatment of diseases by cell therapy, and studies and tests therefor.
  • 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

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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

レトロウイルスベクター製造用の核酸Nucleic acids for retroviral vector production
　本発明は、哺乳動物細胞内で所望の遺伝子を発現するために使用される、レトロウイルスベクターを製造するための核酸構築物、当該核酸構築物からの転写物を含むレトロウイルスベクター、当該ベクター用いた遺伝子導入細胞の生産方法、当該核酸構築物を含む細胞に関する。 The present invention provides a nucleic acid construct for producing a retroviral vector that is used to express a desired gene in mammalian cells, a retroviral vector containing a transcript from the nucleic acid construct, and a gene using the vector. It relates to a method for producing transfected cells and cells containing the nucleic acid construct.
　真核生物への遺伝子導入法としては、ウイルスベクターを用いる方法、裸のＤＮＡをエンドサイトーシスや電気穿孔法、遺伝子銃によって導入する技術などが知られている。ウイルスベクターは、遺伝子治療の分野で基礎から臨床まで幅広く利用されている技術であり、例えばアデノウイルスベクターは標的細胞で目的遺伝子を一過性に大量発現させるのに適しており、レトロウイルスベクターは、宿主染色体への安定な組み込み機能により長期安定発現が可能であり、遺伝病の遺伝子治療の分野で期待されているベクターであり、また、トランスジェニック動物作成の分野でも期待されている技術である。 Known methods for introducing genes into eukaryotes include methods using viral vectors, techniques for introducing naked DNA by endocytosis, electroporation, and gene guns. Viral vectors are a technology that is widely used in the field of gene therapy from basic to clinical. , is capable of long-term stable expression due to its stable integration function into the host chromosome, and is a promising vector in the field of gene therapy for hereditary diseases, and a technology that is also expected in the field of transgenic animal production. .
　レトロウイルスは、ウイルス粒子の構造タンパク質、プロテアーゼ、逆転写酵素、インテグラーゼなどの前駆体タンパク質をコードするｇａｇ／ｐｏｌ遺伝子、エンベロープ糖タンパク質をコードするｅｎｖ遺伝子を有する。これらの遺伝子の両端は、ウイルスゲノムの転写、ウイルスゲノムからの逆転写、逆転写を経て合成された二本鎖ＤＮＡの宿主ＤＮＡへの組み込みに関わる５’ＬＴＲ（Ｌｏｎｇ　Ｔｅｒｍｉｎａｌ　Ｒｅｐｅａｔ）および３’ＬＴＲで挟まれている。レトロウイルス粒子を使用する遺伝子導入システムは、ウイルス粒子の感染力を保ちつつ自己複製を不可能にするため、ｇａｇ／ｐｏｌ遺伝子とｅｎｖ遺伝子を発現する１つまたは２つのパッケージングコンストラクトと、レトロウイルスベクター粒子に取り込まれるＲＮＡをコードするトランスファーベクターに分割されている。トランスファーベクターは、ＬＴＲおよびウイルス粒子へのパッケージングシグナル配列を有し、ｇａｇ／ｐｏｌ遺伝子の大部分とｅｎｖ遺伝子が削除され、例えば、所望の配列が挿入されている。トランスファーベクターの５’ＬＴＲのプロモーター配列によりＲＮＡゲノムが転写され、転写物はパッケージングシグナル配列によりウイルス粒子にパッケージングされる。 Retroviruses have gag/pol genes that encode precursor proteins such as viral particle structural proteins, protease, reverse transcriptase, and integrase, and env genes that encode envelope glycoproteins. Both ends of these genes are 5'LTR (Long Terminal Repeat) and 3'LTR involved in transcription of the viral genome, reverse transcription from the viral genome, and integration of the double-stranded DNA synthesized through the reverse transcription into the host DNA. sandwiched between A gene transfer system using retroviral particles consists of one or two packaging constructs expressing the gag/pol and env genes and a retroviral It is divided into transfer vectors that encode RNAs that are incorporated into vector particles. The transfer vector has the LTR and the packaging signal sequence for the viral particle, has most of the gag/pol genes and the env gene deleted, and has, for example, the desired sequence inserted. The RNA genome is transcribed by the promoter sequence of the 5'LTR of the transfer vector, and the transcript is packaged into virus particles by the packaging signal sequence.
　レトロウイルスの自己複製の可能性をより低減させるため、標的細胞の染色体上でプロモーターとして機能する３’ＬＴＲのプロモーター配列を欠失させた自己不活性化（ＳＩＮ）型のレトロウイルスベクターが開発されている。ＳＩＮ型ベクターでは、所望の遺伝子の転写調節は、パッケージングシグナル配列の３’末端側に位置する内部プロモーターにより行われる。ＳＩＮ型ベクターは内部プロモーターより５’末端側の配列が転写されないため自己複製能を有するウイルス粒子が生成する可能性は極めて低く安全である。これまでに、安全性を高めたベクターが報告されている（非特許文献１、非特許文献２）。 In order to further reduce the possibility of retroviral self-replication, a self-inactivating (SIN) retroviral vector has been developed in which the promoter sequence of the 3'LTR that functions as a promoter on the target cell chromosome is deleted. ing. In SIN-type vectors, transcriptional regulation of the desired gene is performed by an internal promoter located 3' to the packaging signal sequence. Since the SIN vector does not transcribe the sequence on the 5' end side of the internal promoter, the possibility of generating self-replicating viral particles is extremely low and it is safe. So far, vectors with improved safety have been reported (Non-Patent Document 1, Non-Patent Document 2).
　哺乳動物細胞内で所望の遺伝子を発現するために使用される、より安全で、より高効率なベクターの開発が望まれている。 　There is a desire to develop safer and more efficient vectors that can be used to express desired genes in mammalian cells.
　本発明の目的は、より安全かつ効率的に、細胞に外来性の遺伝子を導入して発現させるためのレトロウイルスベクターを提供することにある。 The purpose of the present invention is to provide a retroviral vector for introducing and expressing foreign genes into cells more safely and efficiently.
　本発明者らは上記の課題を解決すべく鋭意努力した結果、導入された遺伝子の発現と安全性を両立できるレトロウイルスベクターを見出し、本発明を完成させた。 As a result of diligent efforts to solve the above problems, the present inventors discovered a retroviral vector that can achieve both expression of the introduced gene and safety, and completed the present invention.
　すなわち本発明を概説すれば、
［１］　５’末端から順に、
（ａ）外来のプロモーター配列を含むレトロウイルス由来の５’ＬＴＲ（Ｌｏｎｇ　Ｔｅｒｍｉｎａｌ　Ｒｅｐｅａｔ）配列、
（ｂ）レトロウイルス由来のパッケージングシグナル配列（ψ）、
（ｃ）１８３～２２７ｂｐの長さのｇａｇタンパク質をコードする核酸に由来する配列、
（ｄ）所望の配列またはマルチクローニングサイト、
（ｅ）レトロウイルス由来の３’ＬＴＲ配列、
の各配列を含む、レトロウイルスベクターを製造するための核酸構築物。
［２］　さらに、Ｘプロテインをコードする核酸配列にフレームシフトを生じる配列またはＸプロテインの翻訳を中断する終止コドンが挿入された転写後調節配列（ＰＲＥ）を含む［１］記載の核酸構築物。
［３］
　ＰＲＥがウッドチャック肝炎ウイルス由来のＰＲＥ（ＷＰＲＥ）である［２］記載の核酸構築物。
［４］
　外来のプロモーター配列がサイトメガロウイルス由来プロモーターである［１］記載の核酸構築物。
［５］　所望の配列が内部プロモーター配列を含む配列である［１］記載の核酸構築物。
［６］　所望の配列がＴ細胞受容体（ＴＣＲ）またはキメラ抗原受容体（ＣＡＲ）をコードする配列を含む［１］記載の核酸構築物。
［７］　所望の配列が２Ａペプチドで連結されたＴＣＲα鎖およびＴＣＲβ鎖をコードする配列を含む［６］記載の核酸構築物。
［８］　ＬＴＲ配列がレンチウイルス由来の配列である［１］記載の核酸構築物。
［９］　３’ＬＴＲ配列が自己不活性化（ＳＩＮ）ＬＴＲ配列である［１］記載の核酸構築物。
［１０］　［１］～［９］のいずれか１項記載の核酸構築物からの転写物を含むレトロウイルスベクター。
［１１］　オンコレトロウイルスまたはレンチウイルス由来の５’ＬＴＲ、パッケージングシグナル配列および３’ＬＴＲを含む［１０］記載のレトロウイルスベクター。
［１２］　レトロウイルス粒子を生成する能力を有する細胞に［１］～［９］のいずれか１項記載の核酸構築物を導入する工程を含む、レトロウイルスベクターの製造方法。
［１３］　［１０］または［１１］に記載のレトロウイルスベクターを細胞に導入する工程を含む遺伝子導入細胞の製造方法。
［１４］　細胞が免疫細胞、免疫細胞に分化しうる細胞またはそれらを含有する細胞集団である［１３］記載の遺伝子導入細胞の製造方法。 That is, if the present invention is outlined,
[1] sequentially from the 5' end,
(a) a retrovirus-derived 5' LTR (Long Terminal Repeat) sequence containing an exogenous promoter sequence;
(b) a packaging signal sequence (ψ) from a retrovirus,
(c) a sequence derived from a nucleic acid encoding a gag protein between 183 and 227 bp in length;
(d) a desired sequence or multiple cloning site;
(e) a 3'LTR sequence from a retrovirus;
A nucleic acid construct for producing a retroviral vector, comprising each sequence of
[2] The nucleic acid construct of [1], further comprising a post-transcriptional regulatory sequence (PRE) inserted with a sequence that causes a frameshift in the nucleic acid sequence encoding the X protein or a termination codon that interrupts the translation of the X protein.
[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 of [1], wherein the desired sequence is a sequence containing an internal promoter sequence.
[6] The nucleic acid construct of [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 of [6], wherein the desired sequence comprises sequences encoding TCRα and TCRβ chains linked by a 2A peptide.
[8] The nucleic acid construct of [1], wherein the LTR sequence is a lentivirus-derived sequence.
[9] The nucleic acid construct of [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 of any one of [1] to [9].
[11] The retroviral vector of [10], comprising a 5'LTR, a packaging signal sequence and a 3'LTR derived from an oncoretrovirus or lentivirus.
[12] A method for producing a retroviral vector, comprising the step of introducing the nucleic acid construct of any one of [1] to [9] into a cell capable of producing retroviral particles.
[13] A method for producing a gene-introduced cell, comprising the step of introducing the retroviral vector of [10] or [11] into a cell.
[14] The method for producing gene-introduced cells according to [13], wherein the cells are immune cells, cells capable of differentiating into immune cells, or cell populations containing them.
　本発明により、効率よく所望の遺伝子を発現させるために使用される、レトロウイルスを製造するための安全性の高い核酸構築物、当該核酸構築物からの転写産物を含むレトロウイルスベクター、当該ベクターを使用する遺伝子導入細胞の製造方法、当該核酸構築物を含む細胞が提供される。これらの核酸構築物、レトロウイルスベクター、細胞は、タンパク質の製造、細胞医療による疾患の治療およびそのための研究、試験に極めて有用である。 According to the present invention, a highly safe nucleic acid construct for producing a retrovirus, a retroviral vector comprising a transcription product from the nucleic acid construct, and the vector are used to efficiently express a desired gene. A method for producing a transgenic cell and a cell containing the nucleic acid construct are provided. These nucleic acid constructs, retroviral vectors, and cells are extremely useful for protein production, treatment of diseases by cell therapy, and studies and tests therefor.
実施例で作製した核酸構築物の構造を示す図である。FIG. 2 is a diagram showing the structure of a nucleic acid construct prepared in Examples. 各核酸構築物を使用して製造したレトロウイルスベクターを感染させた細胞が発現するＺｓＧｒｅｅｎ１タンパク質の蛍光強度の相対値を示す図である。FIG. 2 shows relative fluorescence intensity values of ZsGreen1 protein expressed by cells infected with retroviral vectors produced using respective nucleic acid constructs. 各核酸構築物を使用して製造したレトロウイルス溶液のウイルスタイターの相対値を示す図である。FIG. 3 shows the relative viral titers of retroviral solutions produced using each nucleic acid construct. 実施例で作製した核酸構築物の構造を示す図である。FIG. 2 is a diagram showing the structure of a nucleic acid construct prepared in Examples. 各核酸構築物を使用して製造したレトロウイルス溶液のウイルスタイターを示す図である。FIG. 4 shows the virus titers of retroviral solutions produced using each nucleic acid construct. 実施例で作製した核酸構築物の構造を示す図である。FIG. 2 is a diagram showing the structure of a nucleic acid construct prepared in Examples. 各核酸構築物を使用して製造したレトロウイルス溶液のウイルスタイターを示す図である。FIG. 4 shows the virus titers of retroviral solutions produced using each nucleic acid construct. 各核酸構築物を使用して製造したレトロウイルスベクターを感染させた細胞のち、ＣＡＲタンパク質の発現が陽性である細胞の割合を示す図である。FIG. 2 shows the proportion of cells positive for CAR protein expression among cells infected with retroviral vectors produced using each nucleic acid construct. 各核酸構築物を使用して製造したレトロウイルスベクターを感染させた細胞のゲノムに組み込まれたウイルスコピー数を示す図である。FIG. 2 shows the number of viral copies integrated into the genome of cells infected with retroviral vectors produced using each nucleic acid construct. 各核酸構築物を使用して製造したレトロウイルスベクターを感染させたＣＡＲ発現細胞の細胞障害活性を示す図である。FIG. 3 shows cytotoxic activity of CAR-expressing cells infected with retroviral vectors produced using each nucleic acid construct. 実施例で作製した核酸構築物の構造を示す図である。FIG. 2 is a diagram showing the structure of a nucleic acid construct prepared in Examples. 各核酸構築物を使用して製造したレトロウイルスベクターを感染させた細胞が発現する野生型ＴＣＲタンパク質のＲＮＡ発現量を示す図である。FIG. 2 shows RNA expression levels of wild-type TCR proteins expressed by cells infected with retroviral vectors produced using respective nucleic acid constructs. 各核酸構築物を使用して製造したレトロウイルスベクターを感染させた細胞が発現するコドン変換型ＴＣＲタンパク質のＲＮＡ発現量を示す図である。FIG. 2 shows RNA expression levels of codon-converted TCR proteins expressed by cells infected with retroviral vectors produced using respective nucleic acid constructs.
　本明細書において「核酸構築物」とは、天然には見出されない１つ以上の機能性単位を含むように構築された配列を含む核酸を意味する。核酸は、ＤＮＡおよび／またはＲＮＡでよく、修飾核酸も含み得る。形状としては、環状、直鎖状、二本鎖、一本鎖、染色体外ＤＮＡ分子（プラスミド）、コスミドなどが挙げられる。また、核酸構築物は、遺伝子をコードする核酸配列の他、必要に応じて作動可能に（すなわち、転写や翻訳を制御し得るように）連結された制御配列（例えば、プロモーター）を含む核酸配列を含み得る。この核酸構築物は、必要に応じて他の調節エレメント、機能性配列、リンカーなどを含み得る。 As used herein, the term "nucleic acid construct" means a nucleic acid containing a sequence constructed to contain one or more functional units not found in nature. Nucleic acids can be DNA and/or RNA and can also include modified nucleic acids. Forms include circular, linear, double-stranded, single-stranded, extrachromosomal DNA molecules (plasmids), cosmids, and the like. Nucleic acid constructs also include a nucleic acid sequence encoding a gene, optionally including a control sequence (e.g., promoter) operably linked (i.e., capable of controlling transcription or translation). can contain. The nucleic acid construct may optionally contain other regulatory elements, functional sequences, linkers, and the like.
　本明細書において「ＬＴＲ（Ｌｏｎｇ　Ｔｅｒｍｉｎａｌ　Ｒｅｐｅａｔ）」とは、レトロウイルス、レトロトランスポゾンなどのプロウイルスＤＮＡの両端に繰り返されている１００～１０００塩基対からなる配列をいう。ＬＴＲは、ウイルスゲノムの転写、ウイルスゲノムからの逆転写、逆転写を経て合成された二本鎖ＤＮＡの宿主ＤＮＡへの組み込みに関わるＵ３、ＲおよびＵ５の各領域から構成される。プロウイルス５’末端および３’末端にあるＩＲ配列（ｉｎｖｅｒｔｅｄ　ｒｅｐｅａｔ　ｒｅｇｉｏｎ＝逆方向反復）は４～２０塩基対の長さである。Ｕ３には転写のエンハンサー配列およびプロモーター配列が含まれる。 As used herein, "LTR (Long Terminal Repeat)" refers to a sequence consisting of 100 to 1000 base pairs repeated at both ends of proviral DNA such as retroviruses and retrotransposons. The LTR is composed of U3, R, and U5 regions 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 4-20 base pairs long. U3 contains transcriptional enhancer and promoter sequences.
　本明細書において「パッケージングシグナル配列」は、「プサイ（ｐｓｉ）配列」または「ψ配列」とも表記され、ウイルス粒子の形成においてレトロウイルスＲＮＡ鎖のキャプシド形成およびウイルス粒子へのパッケージングに必要な、非コード性のシス作用性の配列を意味する。例えば、主要なスプライスドナー（ＳＤ）部位の３’側からｇａｇ開始コドンまでの領域、またはＳＤ部位の３’側からｇａｇ遺伝子配列の一部を含む領域である。 As used herein, the “packaging signal sequence” is also referred to as “psi sequence” or “ψ sequence” and is required for encapsidation of retroviral RNA strands and packaging into viral particles in the formation of viral particles. , refers to non-coding cis-acting sequences. For example, the region 3' of the major splice donor (SD) site to the gag initiation codon, or the region 3' of the SD site that includes part of the gag gene sequence.
　本明細書において「所望の配列（ｓｅｑｕｅｎｃｅ　ｏｆ　ｉｎｔｅｒｅｓｔ）」とは、細胞（例えば、細胞の核ゲノムや細胞質）中に一時的もしくは永続的のいずれかで人工的に（人為的な操作により）挿入されることが望まれる外来性の配列を意味する。このような配列には、導入する細胞に対して完全に異種由来または部分的に異種由来である遺伝子配列が含まれ、また、任意の変異を有する遺伝子配列も含まれる。また、その細胞が天然に有している内在性遺伝子と同一の遺伝子配列でもありうる。ここで「天然に」とは、人為的な操作が加えられていない自然の状態であることを意味する。 As used herein, the term "sequence of interest" refers to an artificial (by artificial manipulation) insertion into a cell (e.g., the nuclear genome or cytoplasm of a cell), either transiently or permanently. means an exogenous sequence that is desired to be Such sequences include gene sequences that are wholly or partially heterologous to the cell into which they are introduced, and also include gene sequences with any mutations. It may also be the same gene sequence as the endogenous gene that the cell naturally has. Here, "naturally" means being in a natural state without any artificial manipulation.
　本明細書において「マルチクローニングサイト」は、複数のクローニング用制限酵素部位からなるクラスター配列である。マルチクローニングサイトを構成する塩基配列や包含する制限酵素部位の種類および数については、特に制限はない。 As used herein, a "multicloning site" is a cluster sequence consisting of multiple restriction enzyme sites for cloning. There are no particular restrictions on the nucleotide sequence that constitutes the multicloning site and the type and number of restriction enzyme sites included.
　本明細書において「転写後調節配列（ＰＲＥ：ｐｏｓｔｔｒａｎｓｃｒｉｐｔｉｏｎａｌ　ｒｅｇｕｌａｔｏｒｙ　ｅｌｅｍｅｎｔ）」とは、細胞内で遺伝子から転写されたｍＲＮＡのポリアデニレーションの促進、ｍＲＮＡの核外輸送の促進またはｍＲＮＡの翻訳の活性化に寄与する配列のことを示す。本発明の核酸構築物に含まれる所望の遺伝子の非翻訳領域に挿入することにより、所望の遺伝子のタンパク質レベルの発現が向上する。 As used herein, the term "posttranscriptional regulatory element (PRE)" refers to promoting polyadenylation of mRNA transcribed from a gene in a cell, promoting nuclear export of mRNA, or activating translation of mRNA. indicates a sequence that contributes to By inserting into the untranslated region of the desired gene contained in the nucleic acid construct of the present invention, expression of the desired gene at the protein level is enhanced.
　本明細書において「野生型」とは、天然に存在する供給源から単離された遺伝子または遺伝子産物のうち、集団において最も高頻度で観察されるものを意味する。これは、天然より単離するか、または人為的に作製し得る。一方、「変異型」とは、野生型遺伝子または遺伝子産物と比較した場合に、配列および／または機能的特性が改変された遺伝子または遺伝子産物を指す。変異型遺伝子は、自然に変異が生じることにより、または人為的に遺伝子を修飾して配列を変異させることにより産生される。 As used herein, the term "wild-type" means 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 made artificially. "Mutant", on the other hand, refers to a gene or gene product that has altered sequence and/or functional properties when compared to the wild-type gene or gene product. Mutant genes are produced by spontaneous mutation or by artificially modifying a gene to mutate the sequence.
　本明細書において「Ｔ細胞」とは、Ｔリンパ球とも呼ばれ、免疫応答に関与するリンパ球のうち胸腺に由来する細胞を意味する。Ｔ細胞には、ヘルパーＴ細胞、サプレッサーＴ細胞、制御性Ｔ細胞、ＣＴＬ、ナイーブＴ細胞、メモリーＴ細胞、α鎖とβ鎖のＴＣＲを発現するαβＴ細胞、γ鎖とδ鎖のＴＣＲを発現するγδＴ細胞が含まれる。「Ｔ細胞に分化しうる細胞」としては、生体内においてもしくは人為的な刺激によってT細胞に分化する細胞であれば特に限定はないが、例えば、造血幹細胞、多能性前駆細胞、リンパ系共通前駆細胞、Ｔ細胞前駆細胞等が含まれる。「Ｔ細胞またはＴ細胞に分化しうる細胞を含有する細胞集団」としては、血液（末梢血、臍帯血など）、骨髄液の他、これらより採取、単離、精製、誘導された末梢血単核細胞（ＰＢＭＣ）、血球系細胞、造血幹細胞、臍帯血単核球などを含む細胞集団が例示される。また、Ｔ細胞を含有する血球系細胞由来の種々の細胞集団を本発明に使用できる。これらの細胞は抗ＣＤ３抗体やＩＬ－２などのサイトカインにより生体内（イン・ビボ）や生体外（エクス・ビボ）で活性化されていても良い。これらの細胞は生体から採取されたもの、あるいは生体外での培養を経て得られたもの、例えば生体より得られたＴ細胞集団をそのままもしくは凍結保存したもののいずれも使用することができる。 As used herein, "T cells" are also called T lymphocytes, and mean cells derived from the thymus among lymphocytes involved in immune response. T cells include helper T cells, suppressor T cells, regulatory T cells, CTL, naive T cells, memory T cells, αβ T cells expressing α and β chain TCRs, and γ and δ chain TCRs. It includes γδ T cells that "Cells that can differentiate into T cells" are not particularly limited as long as they are cells that differentiate into T cells in vivo or by artificial stimulation. Included are progenitor cells, T-cell progenitor cells, and the like. The "cell population containing T cells or cells capable of differentiating into T cells" includes blood (peripheral blood, umbilical cord blood, etc.), bone marrow fluid, and peripheral blood collected, isolated, purified, and induced therefrom. Cell populations including nuclear cells (PBMC), hematopoietic cells, hematopoietic stem cells, cord blood mononuclear cells and the like are exemplified. Various cell populations derived from blood lineage cells containing T cells can also be used in the present invention. These cells may be activated in vivo or ex vivo by cytokines such as anti-CD3 antibodies and IL-2. These cells can be either collected from a living body or obtained through in vitro culture, for example, a T cell population obtained from a living body as it is or cryopreserved.
　以下、本発明を具体的に説明する。
（１）本発明の核酸構築物
　本発明の核酸構築物は、５’末端から順に、
（ａ）外来のプロモーター配列を含むレトロウイルス由来の５’ＬＴＲ（Ｌｏｎｇ　Ｔｅｒｍｉｎａｌ　Ｒｅｐｅａｔ）配列、
（ｂ）レトロウイルス由来のパッケージングシグナル配列（ψ）、
（ｃ）１８３～２２７ｂｐの長さのｇａｇタンパク質をコードする核酸に由来する配列、
（ｄ）所望の配列またはマルチクローニングサイト、
（ｅ）レトロウイルス由来の３’ＬＴＲ配列、
の各配列を含む、レトロウイルスベクターを製造するための核酸構築物である。本発明の核酸構築物は、レトロウイルスベクターを製造するために使用することができる。すなわち、レトロウイルス粒子を生成する能力を有する細胞に本発明の核酸構築物を導入することで、核酸構築物からの転写物を含む本発明のレトロウイルスベクターを製造することができる。本発明の核酸構築物は、高いウイルスタイターのレトロウイルスベクターを製造することができる。このレトロウイルスベクターは、所望の配列を細胞に導入することを可能とする。本発明のレトロウイルスベクターにより所望の配列が導入された細胞では、所望の配列の発現効率が高い。 The present invention will be specifically described below.
(1) Nucleic acid construct of the present invention
(a) a retrovirus-derived 5' LTR (Long Terminal Repeat) sequence containing an exogenous promoter sequence;
(b) a packaging signal sequence (ψ) from a retrovirus,
(c) a sequence derived from a nucleic acid encoding a gag protein between 183 and 227 bp in length;
(d) a desired sequence or multiple cloning site;
(e) a 3'LTR sequence from a retrovirus;
is a nucleic acid construct for producing a retroviral vector, comprising each sequence of The nucleic acid constructs of the invention can be used to produce retroviral vectors. That is, by introducing the nucleic acid construct of the present invention into a cell capable of producing retroviral particles, the retroviral vector of the present invention containing a transcript from the nucleic acid construct can be produced. The nucleic acid constructs of the present invention are capable of producing high viral titer retroviral vectors. This retroviral vector allows the desired sequences to be introduced into the cell. Cells into which a desired sequence has been introduced by the retroviral vector of the present invention have a high expression efficiency of the desired sequence.
　レトロウイルスはプラス鎖の一本鎖ＲＮＡのゲノムを持つエンベロープウイルスであり、ウイルスゲノムには主要な要素として、その５’末端より５’ＬＴＲ配列、ＳＤ配列、パッケージングシグナル配列、ｇａｇ遺伝子、ｐｏｌ遺伝子、ＳＡ配列、ｅｎｖ遺伝子、３’ＬＴＲ配列が存在している。後述するレンチウイルスの場合にはこれらの要素に加えて複数のアクセサリー遺伝子が含まれている。このうち、レトロウイルスベクターによる遺伝子導入システムにおいて、レトロウイルスベクターに必須であるのは５’ＬＴＲ配列、パッケージングシグナル配列、３’ＬＴＲ配列であり、本発明の核酸はこれらすべてを備えている。その他のｇａｇ、ｐｏｌ、ｅｎｖ等の遺伝子産物は、これらの遺伝子を保持させたパッケージング細胞より供給され得る。通常、所望の配列はレトロウイルスベクターのパッケージングシグナル配列の３’側、ＳＡ配列を有する場合はパッケージングシグナル配列およびＳＡ配列の３’側に配置される。 A retrovirus is an enveloped virus having a genome of positive-strand single-stranded RNA, and the viral genome includes, from its 5' end, a 5'LTR sequence, an SD sequence, a packaging signal sequence, a gag gene, a pol. Gene, SA sequence, env gene, 3'LTR sequence are present. In the case of lentivirus, which will be described later, a plurality of accessory genes are included in addition to these elements. Among them, in the retroviral vector-based gene transfer system, the 5'LTR sequence, the packaging signal sequence, and the 3'LTR sequence are essential for the retroviral vector, and the nucleic acid of the present invention comprises all of these. Other gene products such as gag, pol, env, etc. can be supplied from packaging cells carrying these genes. Generally, the desired sequence is placed 3' to the packaging signal sequence of the retroviral vector, or 3' to both the packaging signal sequence and the SA sequence if the SA sequence is present.
　本発明の核酸構築物は、１８３～２２７ｂｐの長さのｇａｇタンパク質をコードする核酸に由来する配列を含む。レトロウイルスのパッケージングシグナル配列は部分的にｇａｇ遺伝子の配列を含むため完全に除去することはできないが、前記の長さの配列が効率的なウイルスの製造と所望の配列の発現に有用である。さらに、ウイルスの製造に使用される全長のｇａｇ遺伝子との相同組換えの危険性を低減させることができため、本発明は安全性の高いウイルスベクターの製造方法を提供することができる。例えば、ｇａｇタンパク質をコードする核酸に由来する配列は、１８３ｂｐまたは２２７ｂｐの長さが好適であり、配列番号４または３に記載される配列、またはこの配列に１もしくは数個、例えば１～９個の塩基が置換、欠失、挿入もしくは付加された塩基配列が例示される。 A nucleic acid construct of the present invention comprises a sequence derived from a nucleic acid encoding a gag protein with a length of 183-227 bp. Although the retroviral packaging signal sequence partially includes the sequence of the gag gene and cannot be completely removed, a sequence of this length is useful for efficient virus production and expression of the desired sequence. . Furthermore, since the risk of homologous recombination with the full-length gag gene used for virus production can be reduced, the present invention can provide a method for producing a highly safe viral vector. For example, a sequence derived from a nucleic acid encoding a gag protein is suitably 183 bp or 227 bp in length and is the sequence set forth in SEQ ID NO: 4 or 3, or one or several, such as 1-9 are substituted, deleted, inserted or added.
　本発明の核酸構築物に含まれる（ａ）外来のプロモーター配列を含む５’ＬＴＲ配列、（ｅ）３’ＬＴＲ配列および（ｂ）パッケージングシグナル配列は、レトロウイルス由来の配列で、これら配列を備えたＲＮＡをゲノムとして含むレトロウイルスを産生することが可能な配列であればいずれも使用することができる。レトロウイルスは、オンコレトロウイルスとレンチウイルスのサブクラスを含み、いずれのクラスのウイルス由来の配列も本発明に使用できる。これらの配列は同一のウイルス由来の配列であってもよいが、適切なパッケージング細胞との組合せによりウイルス粒子の形成や導入細胞ゲノムへの組み込みが可能な範囲で異なるウイルス由来の配列を組み合わせて使用してもよい。 The (a) 5'LTR sequence containing the exogenous promoter sequence, (e) the 3'LTR sequence and (b) the packaging signal sequence contained in the nucleic acid construct of the present invention are retrovirus-derived sequences and comprise these sequences. Any sequence capable of producing a retrovirus containing the RNA as its genome can be used. Retroviruses include subclasses of oncoretroviruses and lentiviruses, and sequences from either class of virus can be used in the present invention. These sequences may be sequences derived from the same virus, but sequences derived from different viruses may be combined to the extent that viral particles can be formed and integration into the genome of the transfected cell can be achieved by combination with an appropriate packaging cell. may be used.
　本発明で使用するＬＴＲ配列およびパッケージングシグナル配列には、例えば、オンコレトロウイルスに属するモロニーマウス白血病ウイルス（ＭＭＬＶ）、マウス胚性幹細胞ウイルス（ＭＥＳＶ）、マウス幹細胞ウイルス（ＭＳＣＶ）、骨髄増殖性肉腫ウイルス（ＭＰＳＶ）、脾限局巣形成ウイルス（ＳＦＦＶ）由来の配列が使用できる。オンコレトロウイルス由来のウイルスベクターは、高効率で遺伝子導入可能であるが、ベクターの導入の際に細胞が活発に細胞分裂を行っている必要がある。これらのオンコレトロウイルスベクターは多数の文献で説明されている［例えば米国特許第５，２１９，７４０号公報、米国特許第６，２０７，４５３号公報、米国特許第５，２１９，７４０号公報、バイオテクニークス（ＢｉｏＴｅｃｈｎｉｑｕｅｓ）第７巻、第９８０－９９０頁（１９８９）、ヒューマン　ジーン　セラピー（Ｈｕｍａｎ　Ｇｅｎｅ　Ｔｈｅｒａｐｙ）、第１巻、第５－１４頁（１９９０）、ヴイロロジー（Ｖｉｒｏｌｏｇｙ）、第１８０巻、第８４９－８５２頁（１９９１）、米国科学アカデミー紀要（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ）、第９０巻、第８０３３－８０３７頁（１９９３）、ならびにカレント　オピニオン　イン　ジェネティックス　アンド　デベロップメント（Ｃｕｒ．Ｏｐｉｎ．Ｇｅｎｅｔ．Ｄｅｖｅｌｏｐ．）、第３巻、第１０２－１０９頁（１９９３）］。 LTR sequences and packaging signal sequences used in the present invention include, for example, Moloney murine leukemia virus (MMLV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), myeloproliferative sarcoma, which belongs to the oncoretroviruses. Sequences from viruses (MPSV), splenic focal-focal virus (SFFV) can be used. Oncoretrovirus-derived viral vectors are capable of highly efficient gene transfer, but cells must be actively undergoing cell division at the time of vector transfer. These oncoretroviral vectors have been described in numerous publications [e.g., US Pat. No. 5,219,740, US Pat. No. 6,207,453, US Pat. 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. Develop., Vol. 3, pp. 102-109 (1993)].
　また、本発明で使用するＬＴＲ配列およびパッケージングシグナル配列には、例えば、レンチウイルスに属するヒト免疫不全ウイルス（ＨＩＶ－１、ＨＩＶ－２）、サル免疫不全ウイルス（ＳＩＶ）、ネコ免疫不全ウイルス（ＦＩＶ）、ウマ伝染性貧血ウイルス（ＥＩＡＶ）、ヤギ関節炎脳炎ウイルス（ＣＡＥＶ）由来の配列が使用できる。レンチウイルスベクターは、遺伝子を導入する細胞の有糸分裂に関係なく核内のゲノムに遺伝子を導入ことができる。レンチウイルスベクターも多数の文献で説明されている［例えば、ジャーナル　オブ　ヴイロロジー（Ｊ．Ｖｉｒｏｌｏｇｙ）、第７２巻、第８４６３－８４７１頁（１９９８）］。スプマウイルス属（例えば、泡沫状ウイルス）のような他のレトロウイルスのグループも、分裂していない細胞に効率的に形質導入することができる。 In addition, the LTR sequence and packaging signal sequence used in the present invention include, for example, human immunodeficiency virus (HIV-1, HIV-2) belonging to lentiviruses, simian immunodeficiency virus (SIV), feline immunodeficiency virus ( FIV), Equine Infectious Anemia Virus (EIAV), Captive Arthritis Encephalitis Virus (CAEV) can be used. Lentiviral vectors can introduce genes into the genome in the nucleus regardless of mitosis of the cells into which they are introduced. Lentiviral vectors have also been described in numerous publications [eg, J. Virology, 72:8463-8471 (1998)]. Other groups of retroviruses, such as spumaviruses (eg, foamy viruses), can also efficiently transduce non-dividing cells.
　ＬＴＲは機能的に５’末端よりＵ３、Ｒ、Ｕ５の３つの領域に区分される。Ｕ３領域にはエンハンサー／プロモーター活性があり、宿主細胞のＲＮＡポリメラーゼＩＩによって、ウイルスゲノムは５’ＬＴＲ配列のＲ領域から３’ＬＴＲ配列のＲ領域までが転写される。本発明で使用する５’ＬＴＲ配列は、ＬＴＲが由来するウイルスに対して外来性のエンハンサー／プロモーターを含む。本発明の一態様として、５’ＬＴＲのＵ３領域が外来性エンハンサー／プロモーターに置換されている。さらに、３’ＬＴＲ配列のＵ３領域を、そのＬＴＲ配列が由来するウイルス以外に由来するエンハンサー／プロモーターに置換したＬＴＲ配列も本発明に使用できる。置換する外来性のエンハンサー／プロモーターは、ウイルスまたは哺乳動物由来の配列を使用することができ、構成的、誘導性または組織特異的なエンハンサー／プロモーターが使用できる。例えばヒトサイトメガロウイルス（ＨＣＭＶ）　ｉｍｍｅｄｉａｔｅ　ｅａｒｌｙ、モロニーマウス肉腫ウイルス（ＭＭＳＶ）、マウス幹細胞ウイルス（ＭＳＣＶ）、ラウス肉腫ウイルス（ＲＳＶ）、脾臓フォーカス形成ウイルス（ＳＦＦＶ）等のウイルス由来エンハンサー／プロモーターや、βアクチン、グロビン、エラスターゼ、アルブミン、α－フェトプロテインおよびインスリン遺伝子の哺乳動物由来エンハンサー／プロモーターが使用できる。ここで、エンハンサー／プロモーターの語は、エンハンサー領域および／またはプロモーター領域を含む配列を指す。一般に、エンハンサー領域およびプロモーター領域を総称してプロモーターと称することがある。また、エンハンサー領域と区別するため、プロモーター領域をコアプロモーターと称することがある。本発明には、エンハンサー領域および／またはプロモーター領域のいずれも使用することができる。本発明の一態様として、外来のプロモーター配列を含む５’ＬＴＲ配列は、配列番号７に記載される配列、またはこの配列に１もしくは数個、例えば１～９個の塩基が置換、欠失、挿入もしくは付加された塩基配列を含む。 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 RNA polymerase II of the host cell. The 5'LTR sequences used in the present invention contain enhancers/promoters that are foreign to the virus from which the LTR is derived. In one aspect of the invention, the U3 region of the 5'LTR is replaced with an exogenous enhancer/promoter. Furthermore, an LTR sequence in which the U3 region of the 3'LTR sequence is replaced with an enhancer/promoter derived from a virus other than the virus from which the LTR sequence is derived can also be used in the present invention. The exogenous enhancer/promoter to replace can be of viral or mammalian origin and can be constitutive, inducible or tissue-specific. For example, virus-derived enhancers/promoters such as human cytomegalovirus (HCMV) immediate early, Moloney murine sarcoma virus (MMSV), mouse stem cell virus (MSCV), Rous sarcoma virus (RSV), spleen focus-forming virus (SFFV), etc. Mammalian enhancers/promoters of actin, globin, elastase, albumin, α-fetoprotein and insulin genes can be used. As used herein, the term enhancer/promoter refers to sequences comprising enhancer and/or promoter regions. In general, enhancer regions and promoter regions are sometimes collectively referred to as promoters. In order to distinguish from the enhancer region, the promoter region is sometimes called a core promoter. Either enhancer regions and/or promoter regions can be used in the present invention. As one aspect of the present invention, the 5' LTR sequence containing the foreign promoter sequence is the sequence set forth in SEQ ID NO: 7, or one or several, for example, 1 to 9, base substitutions, deletions, or substitutions in this sequence. Including inserted or added base sequences.
　本発明で使用する３’ＬＴＲ配列は、Ｕ３領域に変異を導入してエンハンサー／プロモーター活性を欠失させた配列を使用することができる。これにより、この核酸構築物からの転写物を含むウイルスベクターが細胞に感染した際、ウイルスゲノムが細胞染色体に組み込まれて形成されたプロウイルスでは、Ｒ領域からの転写が抑制される。このように３’ＬＴＲ配列のＵ３領域を変異させたレトロウイルスベクターを自己不活性化（ＳＩＮ）型ベクターと呼ぶ。３’ＬＴＲ配列への変異の導入は、塩基の置換または欠失により実施される。ＳＩＮ型ベクターはプロウイルスでのＬＴＲのＲ領域からの転写が抑制されているため、所望の配列を発現させるためのプロモーター配列をＬＴＲとは別に配置する必要がある。さらに、複数の所望の配列を発現させる場合にも、ＬＴＲとは別のプロモーター配列を配置する。このような、５’ＬＴＲおよび３’ＬＴＲの間に存在するプロモーター配列を内部プロモーター配列と言うことがある。内部プロモーター配列は、ウイルス由来または哺乳動物遺伝子由来のプロモーター配列が使用できる。ウイルス由来のプロモーター配列を使用する場合は、５’ＬＴＲ、３’ＬＴＲまたはパッケージングシグナル配列が由来するウイルスと同一のウイルス由来の配列または異なるウイルス由来の配列を使用できる。例えば、レトロウイルスのＬＴＲのＵ３領域由来のプロモーター配列、例えば、マウス幹細胞ウイルス（ＭＳＣＶ）のＬＴＲのＵ３領域由来のプロモーター配列等が使用できる。また、内部プロモーター配列は、前段落の５’ＬＴＲ配列のＵ３領域を置換する外来性プロモーターとして例示する配列が使用できる。ウイルス由来のプロモーター配列としては、上記の他、ＳＶ４０プロモーター、ＣＭＶプロモーター等の配列を使用することができる。さらに、哺乳動物細胞で機能するプロモーターである、ホスホグリセリン酸キナーゼ（ＰＧＫ）プロモーター、ポリペプチド鎖伸長因子（ＥＦ１-α）プロモーター、β－アクチンプロモーター、ＣＡＧプロモーター等の配列も使用することができる。なお、内部プロモーターは、（ｄ）の上流（すなわち、５’側）に配置すればよく、（ａ）と（ｄ）の間、好適には（ｂ）と（ｄ）の間に配置することができる。 For the 3'LTR sequence used in the present invention, a sequence in which enhancer/promoter activity is deleted by introducing mutations into the U3 region can be used. As a result, when cells are infected with a viral vector containing a transcript from this nucleic acid construct, transcription from the R region is suppressed in a provirus formed by integrating the viral genome into the cell chromosome. Such a retroviral vector in which the U3 region of the 3'LTR sequence is mutated is called a self-inactivating (SIN) vector. Mutations are introduced into the 3'LTR sequence by base substitution or deletion. Since transcription from the R region of the LTR in the provirus is suppressed in the SIN-type vector, it is necessary to place a promoter sequence separately from the LTR for expressing the desired sequence. Furthermore, when expressing multiple desired sequences, a promoter sequence separate from the LTR is arranged. Such a promoter sequence existing between the 5'LTR and the 3'LTR is sometimes referred to as an internal promoter sequence. As the internal promoter sequence, promoter sequences derived from viruses or mammalian genes can be used. If a viral promoter sequence is used, it can be 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 the LTR of a retrovirus, such as a promoter sequence derived from the U3 region of the LTR of a mouse stem cell virus (MSCV), or the like can be used. As the internal promoter sequence, the sequence exemplified as the exogenous promoter that replaces the U3 region of the 5'LTR sequence in the preceding paragraph can be used. As a virus-derived promoter sequence, in addition to the above, sequences such as SV40 promoter and CMV promoter can be used. Furthermore, sequences such as phosphoglycerate kinase (PGK) promoter, polypeptide chain elongation factor (EF1-α) promoter, β-actin promoter, and CAG promoter, which are promoters that function in mammalian cells, can also be used. The internal promoter may be placed upstream of (d) (that is, on the 5' side), and should be placed between (a) and (d), preferably between (b) and (d). can be done.
　本発明の核酸構築物は、ＳＤ配列および／またはＳＡ配列を含んでいても良い。これらの配列は、ＬＴＲに対して外来性のＳＤ配列および／またはＳＡ配列、または、内部プロモーター配列に対して外来性のＳＤ配列および／またはＳＡ配列が使用できる。また、ＳＤ配列およびＳＡ配列は、互いに異なる由来の配列であってもよい。例えば、ｓｉｍｉａｎ　ｖｉｒｕｓ（ＳＶ）４０の１６Ｓ　ＲＮＡ、ＨＣＭＶのｉｍｍｅｄｉａｔｅ　ｅａｒｌｙ　ＲＮＡ、ヒトのｈＥＦ１α遺伝子由来のＳＤ配列およびＳＡ配列が使用できる［米国科学アカデミー紀要、第９５巻、第１号、第２１９－２２３頁（１９９８）］。また、コンセンサス配列に変異を導入し、スプライシング活性を強化または抑制されたＳＤ配列またはＳＡ配列も本発明の核酸構築物に使用できる。 The nucleic acid construct of the present invention may contain an SD sequence and/or an SA sequence. These sequences can be SD and/or SA sequences exogenous to the LTR, or SD and/or SA sequences exogenous to the internal promoter sequence. Moreover, the SD sequence and the SA sequence may be sequences of different origins. For example, simian virus (SV) 40 16S RNA, HCMV immediate early RNA, human hEF1α gene-derived SD and SA sequences can be used [Proceedings of the National Academy of Sciences, Vol. 95, No. 1, 219-223. (1998)]. Moreover, the SD sequence or SA sequence, in which the consensus sequence is mutated to enhance or suppress the splicing activity, can also be used in the nucleic acid construct of the present invention.
　本発明の核酸構築物に含まれる（ｄ）の所望の配列は、製造するベクターを導入する細胞で発現させたい配列である。当該配列には、例えばタンパク質をコードする配列、ｔＲＮＡやｍｉＲＮＡ等の細胞内で機能するＲＮＡをコードする配列が含まれる。また、所望の配列を連結するための制限酵素認識配列を複数個並べた配列（マルチクローニングサイト）を配列（ｄ）として配置した核酸構築物を製造し、その後マルチクローニングサイトを利用して所望の配列を挿入しても良い。このような、所望の配列の代わりにマルチクローニングサイトを有する核酸構築物も本発明の核酸構築物に含まれる。 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. Such sequences include, for example, sequences encoding proteins, and sequences encoding RNAs that function in cells, such as tRNAs and miRNAs. Alternatively, a nucleic acid construct is prepared in which a sequence (d) in which a plurality of restriction enzyme recognition sequences for ligating desired sequences are arranged (multicloning site) is arranged, and then the desired sequence is obtained using the multicloning site. may be inserted. Such nucleic acid constructs having multiple cloning sites in place of desired sequences are also included in the nucleic acid constructs of the present invention.
　前記の所望の配列は、疾病の予防や治療を目的とするものであってもよい。生体において有害な遺伝子産物の転写や発現を抑制するのに有用な配列（例えばｓｉＲＮＡをコードする配列）、生体で欠失もしくは機能を失っているタンパク質等を補充するための、当該タンパク質をコードする配列、細胞の有する機能を改変または強化することのできる配列等が例示される。本発明の核酸構築物により外来の配列を導入した細胞を生体に導入する遺伝子治療が本発明により提供される。例えば、ＩＬ－２受容体γ鎖をコードする配列（Ｘ連鎖重症複合免疫不全症）、βグロビンをコードする配列（βサラセミア）、アデノシンデアミナーゼ（ＡＤＡ）をコードする配列（ＡＤＡ欠損症）、血液凝固因子をコードする配列（血友病）、抗原を認識する受容体をコードする配列（癌やウイルス感染症）を使用する遺伝子治療が例示される。 The desired sequence may be for the purpose of disease prevention or treatment. A sequence useful for suppressing the transcription or expression of a gene product that is harmful in vivo (for example, a sequence encoding siRNA), or a protein that is used to replenish a protein that has been deleted or has lost its function in vivo. Sequences, sequences capable of modifying or enhancing functions of cells, and the like are exemplified. The present invention provides gene therapy in which cells into which a foreign sequence has been introduced by the nucleic acid construct of the present invention are introduced into a living body. For example, sequences encoding the IL-2 receptor γ chain (X-linked severe combined immunodeficiency), sequences encoding β-globin (β-thalassemia), sequences encoding adenosine deaminase (ADA) (ADA deficiency), blood Gene therapy using sequences encoding clotting factors (hemophilia), sequences encoding receptors that recognize antigens (cancer and viral infections) are exemplified.
　本発明の一態様として、本発明の核酸構築物に含まれる所望の配列はオリゴマータンパク質をコードする配列である。オリゴマータンパク質には、構造タンパク質、酵素、転写因子、レセプター、抗体が含まれる。また、本発明において、オリゴマータンパク質は細胞表面タンパク質（膜タンパク質）であってもよく、実施例に例示された抗原認識レセプター、例えばＴ細胞レセプター（Ｔ細胞受容体：ＴＣＲ）をコードする配列が所望の配列として好適である。 In one aspect 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. Further, in the present invention, the oligomer protein may be a cell surface protein (membrane protein), and a sequence encoding an antigen recognition receptor exemplified in the Examples, such as a T cell receptor (T cell receptor: TCR) is desirable. is suitable as an array of
　本発明の一態様として、本発明の核酸構築物に含まれる所望の配列はキメラ抗原受容体（ＣＡＲ）をコードする配列である。代表的なＣＡＲの構造は、生体から排除したい標的細胞、例えば腫瘍細胞の表面抗原を認識する単鎖抗体（ｓｉｎｇｌｅ　ｃｈａｉｎ　ｖａｒｉａｂｌｅ　ｆｒａｇｍｅｎｔ：ｓｃＦｖ）、膜貫通ドメインとＴ細胞を活性化させる細胞内ドメインから構成される。細胞内ドメインとしては、ＴＣＲ複合体ＣＤ３ζの細胞内ドメインが好適に使用されている。このような構成のＣＡＲは第一世代ＣＡＲと呼ばれている。単鎖抗体部分の遺伝子は、例えば標的とする抗原を認識するモノクローナル抗体を産生するハイブリドーマから単離することができる。ＣＡＲを発現するＴ細胞は、腫瘍細胞上の主要組織適合抗原クラスＩの発現とは無関係に標的細胞の表面抗原を直接認識し、同時にＴ細胞を活性化することで、効率よく標的細胞を殺傷することが可能である。 As one aspect 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 consists of a single chain antibody (single chain variable fragment: scFv) that recognizes surface antigens of target cells to be eliminated from the body, for example, tumor cells, a transmembrane domain, and an intracellular domain that activates T cells. Configured. As the intracellular domain, the intracellular domain of the TCR complex CD3zeta is preferably used. A CAR with such a configuration is called a first generation CAR. The genes for single-chain antibody portions can be isolated, for example, from hybridomas that produce monoclonal antibodies that recognize the target antigen. CAR-expressing T cells directly recognize the surface antigens of target cells, irrespective of the expression of major histocompatibility class I antigens on tumor cells, and simultaneously activate T cells to efficiently kill target cells. It is possible to
　第一世代ＣＡＲのＴ細胞活性化能を増強する目的で、Ｔ細胞の共刺激分子の細胞内ドメインを連結した第二世代ＣＡＲが開発されている。Ｔ細胞の共刺激分子としては、ＣＤ２８、腫瘍壊死因子（ＴＮＦ）受容体スーパーファミリーであるＣＤ１３７（４－１ＢＢ）またはＣＤ１３４（ＯＸ４０）の細胞内ドメインが好適に使用されている。さらなる改良型として、これらの共刺激分子の細胞内ドメインをタンデムに連結した第三世代ＣＡＲも開発され、様々な腫瘍抗原を標的とした多くのＣＡＲ分子が報告されている。本発明の核酸構築物は、所望の配列として、いずれのＣＡＲをコードする配列も含み得る。 With the aim of enhancing the ability of first-generation CARs to activate T cells, second-generation CARs linked to the intracellular domains of T-cell co-stimulatory molecules have been developed. As co-stimulatory molecules for T cells, CD28, the intracellular domain of CD137 (4-1BB) or CD134 (OX40) of the tumor necrosis factor (TNF) receptor superfamily is preferably used. As a further improvement, third-generation CARs in which the intracellular domains of these co-stimulatory molecules are linked in tandem have also been developed, and many CAR molecules targeting various tumor antigens have been reported. The nucleic acid constructs of the invention can contain sequences encoding any CAR as desired sequences.
　本発明の核酸構築物は転写後調節配列（ＰＲＥ）を含んでも良く、所望の配列の発現を増強させるのに有用である。ＰＲＥは核酸構築物からの転写産物のイントロン内に配置され、レトロウイルスの生活環において、スプライシングにより除去され得る。スプライシングにより除去されないＰＲＥの例は、単純ヘルペスウイルスの転写後処理エレメント、Ｂ型肝炎ウイルス（ＨＰＲＥ）およびウッドチャック肝炎ウイルス（ＷＰＲＥ）の転写後調節配列である。ＰＲＥ配列には発がん性が指摘されているＸプロテインのコード領域が含まれる。本発明の核酸構築物は、ＰＲＥ配列からのＸプロテインの発現が抑制された変異配列も使用できる。例えば、Ｘプロテインをコードする核酸配列にフレームシフトを生じる配列またはＸプロテインの翻訳を中断する終止コドンが挿入されたＰＲＥ配列が使用できる。本発明の一態様として、Ｘプロテインの開始コドンＡＴＧのＡを１位として、６位と７位の間にフレームシフトを生じる１塩基または２塩基の挿入塩基を含むＰＲＥ配列が使用できる。また、７位から９位を終止コドン（例えばＴＡＡ）に置換したＰＲＥ配列が使用できる。本発明にはＷＰＲＥが好ましい。ＷＰＲＥは、米国特許第６，１３６，５９７号公報または米国特許第７，４１９，８２９号公報を参照することができる。本発明の一態様として、ＷＰＲＥ２（配列番号８）またはＷＰＲＥ３（配列番号９）、またはこの配列に１もしくは数個、例えば１～９個の塩基が置換、欠失、挿入もしくは付加された塩基配列が使用できる。本発明の一態様として、（ｄ）所望の配列またはマルチクローニングサイト、と（ｅ）レトロウイルス由来の３’ＬＴＲ配列の間にＰＲＥ配列が配置された核酸構築物が例示される。 The nucleic acid constructs of the present invention may contain post-transcriptional regulatory sequences (PREs), which are useful for enhancing expression of desired sequences. 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 are the post-transcriptional processing elements of herpes simplex virus, the post-transcriptional regulatory sequences of hepatitis B virus (HPRE) and woodchuck hepatitis virus (WPRE). The PRE sequence contains the X protein coding region, which has been pointed out to be carcinogenic. Nucleic acid constructs of the present invention can also use mutant sequences in which the expression of the X protein from the PRE sequence is suppressed. For example, a PRE sequence with an inserted stop codon that causes a frameshift in the nucleic acid sequence encoding the X protein or a stop codon that interrupts translation of the X protein can be used. As one aspect 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 initiation codon ATG of the X protein as position 1 can be used. Also, a PRE sequence in which positions 7 to 9 are replaced with stop codons (eg, TAA) can be used. WPRE is preferred for the present invention. WPRE can be referred to US Pat. No. 6,136,597 or US Pat. No. 7,419,829. As one aspect of the present invention, WPRE2 (SEQ ID NO: 8) or WPRE3 (SEQ ID NO: 9), or a nucleotide sequence in which one or several, for example 1 to 9, bases are substituted, deleted, inserted or added to this sequence can be used. One aspect of the present invention is a nucleic acid construct in which a PRE sequence is positioned between (d) a desired sequence or multiple cloning site and (e) a 3'LTR sequence derived from a retrovirus.
　本発明の核酸構築物は、Ｒｅｖ応答エレメント（ＲＲＥ）配列および／またはセントラルポリプリントラクト（ｃＰＰＴ）配列を含み得る。ＲＲＥはＨＩＶ　ＮＬ４－３ゲノム（ＧｅｎＢａｎｋアクセション番号：ＡＦ００３８８７）の７６２２～８４５９位に位置するようなＲＲＥ、ＨＩＶの他の株または他のレトロウイルスに由来するＲＲＥが例示されるが、これらに限らない。また、ｃＰＰＴはレンチウイルスゲノムのほぼ中央に存在する約１５塩基の配列であり、レンチウイルスゲノムＲＮＡからの二本鎖ＤＮＡ合成の過程でプラス鎖ＤＮＡ合成のためのプライマー結合部位として働く配列である。レンチウイルスのＲＮＡゲノムが逆転写される際、セントラル終結配列（ＣＴＳ：ｃｅｎｔｒａｌ　ｔｅｒｍｉｎａｔｉｏｎ　ｓｅｑｕｅｎｃｅ）と共に機能し、ＤＮＡフラップと呼ばれる３本鎖構造を形成する。なお、本発明の核酸構築物において、両配列の配置には特に限定はなく、５’末端からｃＰＰＴ、ＲＲＥの順およびＲＲＥ、ｃＰＰＴの順のいずれの配置でもよい。 The nucleic acid constructs of the present invention may contain Rev response element (RRE) sequences and/or central polypurine tract (cPPT) sequences. Examples of RRE include, but are not limited to, RREs such as those located at positions 7622-8459 of the HIV NL4-3 genome (GenBank Accession Number: AF003887), RREs derived from other strains of HIV or other retroviruses. do not have. In addition, cPPT is a sequence of about 15 bases that exists almost in the center of the lentiviral genome, and serves as a primer binding site for synthesizing plus-strand DNA in the process of synthesizing double-stranded DNA from lentiviral genomic RNA. . When the lentiviral RNA genome is reverse transcribed, it works 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 both sequences is not particularly limited, and may be arranged in the order of cPPT and RRE or the order of RRE and cPPT from the 5' end.
　本発明は、レトロウイルス粒子を生成する能力を有する細胞に本発明の核酸構築物を導入する工程を含む、レトロウイルスベクター（粒子）の製造方法を含む。核酸構築物は、安定的に細胞内で効果を発揮しうるように、適切なベクター、例えばプラスミドベクター、レトロウイルス以外のウイルスベクターまたはトランスポゾンベクターで細胞に導入され得る。さらに、細胞の染色体ＤＮＡ上にゲノム編集技術を使用して組み込まれ得る。また、この核酸構築物は直接細胞に導入され得る。核酸構築物を直接またはプラスミドベクターで細胞に導入するには、リポソーム、リガンド－ポリリジンなどの担体を使用する方法、リン酸カルシウム法、エレクトロポレーション法、パーティクルガン法などを使用することができる。前記ウイルスベクターには特に限定はなく、通常、遺伝子導入方法に使用される公知のウイルスベクター、例えば、アデノウイルスベクター、アデノ随伴ウイルスベクター、シミアンウイルスベクター、ワクシニアウイルスベクター、麻疹ウイルスベクターまたはセンダイウイルスベクターなどが使用される。 The present invention includes a method for producing a retroviral vector (particle), which includes the step of introducing the nucleic acid construct of the present invention into a cell capable of producing retroviral particles. A nucleic acid construct can be introduced into cells using a suitable vector, such as a plasmid vector, a viral vector other than a retrovirus, or a transposon vector, so that it can stably exert its effect in the cell. Additionally, it can be integrated onto the chromosomal DNA of the cell using genome editing techniques. Alternatively, the nucleic acid construct can be directly introduced into cells. Methods using carriers such as liposomes and ligand-polylysine, calcium phosphate methods, electroporation methods, particle gun methods, and the like can be used to introduce nucleic acid constructs into cells directly or with a plasmid vector. The viral vector is not particularly limited, and is usually a known viral vector used in gene transfer methods, such as adenovirus vector, adeno-associated virus vector, simian virus vector, vaccinia virus vector, measles virus vector or Sendai virus vector. etc. are used.
　レトロウイルス粒子を生成する能力を有する細胞としては、例えば公知のパッケージング細胞を使用することができる。核酸構築物が有しているＬＴＲ配列およびパッケージングシグナル配列に基づいて適切なパッケージング細胞を選択し、前記の核酸構築物を導入してレトロウイルス産生細胞を作製し、レトロウイルスベクター（粒子）を調製することができる。また、トランスフェクション効率の高い細胞（２９３細胞や２９３Ｔ細胞等）に、本発明の核酸構築物と、レトロウイルス粒子産生に必要なコンポーネント（ｇａｇ遺伝子、ｐｏｌ遺伝子、ｅｎｖ遺伝子やその他のアクセサリー遺伝子）をコードする核酸を同時にまたは随時導入し、この細胞をレトロウイルス産生細胞として適切な培地で培養して、レトロウイルス粒子を製造することができる。産生されたレトロウイルス粒子には、本発明の核酸構築物からの転写物が含まれる。このレトロウイルス粒子は、所望の配列を細胞に導入するためのレトロウイルスベクターとして本発明に含まれる。パッケージング細胞としては、例えばＰＧ１３（ＡＴＣＣ　ＣＲＬ－１０６８６）、ＰＡ３１７（ＡＴＣＣ　ＣＲＬ－９０７８）、等が例示される。パッケージング細胞やその他の細胞、レトロウイルス作製用のプラスミド（パッケージングプラスミドと呼ばれる）を含むキット等が各社より広く市販されており、これらを本発明の方法に使用することができる。 As cells capable of producing retroviral particles, for example, known packaging cells can be used. An appropriate packaging cell is selected based on the LTR sequence and packaging signal sequence possessed by the nucleic acid construct, the nucleic acid construct is introduced into the retrovirus-producing cell, and a retroviral vector (particle) is prepared. can do. Alternatively, cells with high transfection efficiency (293 cells, 293T cells, etc.) may be encoded with the nucleic acid construct of the present invention and components (gag gene, pol gene, env gene and other accessory genes) necessary for retroviral particle production. The retroviral particles can be produced by simultaneously or optionally introducing the nucleic acids that are capable of producing retroviral particles and culturing the cells in a suitable medium as retroviral producer cells. The retroviral particles produced contain transcripts from the nucleic acid constructs of the invention. This retroviral particle is included in the present invention as a retroviral vector for introducing desired sequences into cells. Examples of packaging cells include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), and the like. Packaging cells, other cells, kits containing retrovirus-producing plasmids (referred to as packaging plasmids), and the like are widely commercially available from various companies, and these can be used in the method of the present invention.
　本発明には、当該レトロウイルスベクターのゲノムが由来するものとは異種のウイルス由来のエンベロープタンパクを発現するパッケージング細胞、または前記エンベロープタンパクをコードする配列を含むパッケージングプラスミドを使用することにより、シュードタイプ（ｐｓｅｕｄｏｔｙｐｅｄ）のレトロウイルスを製造することができる。例えばＭＭＬＶ、テナガザル白血病ウイルス（ＧａＬＶ）、水泡性口内炎ウイルス（ＶＳＶ）、ネコ内在性ウイルス（ｆｅｌｉｎｅ　ｅｎｄｏｇｅｎｏｕｓ　ｖｉｒｕｓ）由来のエンベロープやエンベロープとして機能しうるタンパク質をコードするパッケージングプラスミドを使用することができる。さらに、糖鎖合成に関与する酵素遺伝子などを導入したパッケージング細胞を使用し、糖鎖修飾を受けたタンパクをその表面に有するレトロウイルスベクターを製造することができる。 In the present invention, by using a packaging cell expressing an envelope protein derived from a virus heterologous to that from which the genome of the retroviral vector is derived, or a packaging plasmid containing a sequence encoding the envelope protein, Pseudotyped retroviruses can be produced. For example, packaging plasmids encoding envelopes derived from MMLV, gibbon leukemia virus (GaLV), vesicular stomatitis virus (VSV), feline endogenous virus or proteins capable of functioning as envelopes can be used. Furthermore, retroviral vectors having sugar chain-modified proteins on their surfaces can be produced using packaging cells into which enzyme genes involved in sugar chain synthesis have been introduced.
　上記の操作により作成されたレトロウイルス産生細胞を培養した後、細胞培養物を遠心分離して上清を回収し、適切なろ過操作により夾雑物を除去してレトロウイルス粒子を得ることができる。この粗精製レトロウイルス粒子をそのまま細胞に接触させて遺伝子導入を行うこともできるが、公知の精製操作を経てより純度の高いレトロウイルス粒子を調製し、遺伝子導入操作に供してもよい。 After culturing the retrovirus-producing cells created by the above operation, the cell culture is centrifuged to collect the supernatant, and contaminants can be removed by appropriate filtration to obtain retrovirus particles. Gene transfer can be performed by directly contacting the crude retroviral particles with cells, but retroviral particles of higher purity may be prepared through known purification procedures and subjected to gene transfer procedures.
　本発明は、本発明のレトロウイルスベクターを有効成分として、薬学的に許容できる賦形剤と共に含む組成物を提供する。薬学的に許容できる賦形剤は、当業者にはよく知られており、例えば、リン酸緩衝生理食塩水（例えば、０．０１Ｍリン酸塩、０．１３８Ｍ　ＮａＣｌ、０．００２７Ｍ　ＫＣｌ、ｐＨ７．４）、塩酸塩、臭化水素酸塩、リン酸塩、硫酸塩などの鉱酸塩を含有する水溶液、生理食塩液、グリコールまたはエタノールなどの溶液および酢酸塩、プロピオン酸塩、マロン酸塩、安息香酸塩などの有機酸の塩を含む。湿潤剤または乳化剤などの補助剤、およびｐＨ緩衝剤も使用することができる。薬学的に許容できる賦形剤としては、Ｒｅｍｉｎｇｔｏｎ’ｓ　Ｐｈａｒｍａｃｅｕｔｉｃａｌ　Ｓｃｉｅｎｃｅｓ（Ｍａｃｋ　Ｐｕｂ．Ｃｏ．、Ｎ．Ｊ．１９９１）（出典明示により本明細書の一部とする）に記載されるものを適宜使用できる。組成物は、非経口投与、例えば、注射または注入に適した公知の形態とすることができる。さらに、懸濁化剤、保存剤、安定化剤および／または分散剤などの製剤補助剤、保存中の有効期限を延ばすために保存剤を使用することができる。組成物は、使用前に適切な無菌の液体により再構成するための乾燥形態であってもよい。 The present invention provides a composition comprising the 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 (eg, 0.01 M phosphate, 0.138 M NaCl, 0.0027 M KCl, pH 7.0). 4), aqueous solutions containing mineral salts such as hydrochlorides, hydrobromides, phosphates, sulfates, physiological saline, solutions such as glycol or ethanol, and acetates, propionates, malonates, Contains salts of organic acids such as benzoate. Adjuvants such as wetting or emulsifying agents, and pH buffering agents can also be used. As pharmaceutically acceptable excipients, those described in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991) (incorporated herein by reference) are used as appropriate. can. The composition can be in any known form suitable for parenteral administration, eg injection or infusion. Additionally, formulation aids such as suspending, preserving, stabilizing and/or dispersing agents, and preservatives can be used to extend shelf life during storage. The composition may be in dry form for reconstitution with a suitable sterile liquid before use.
（２）本発明の遺伝子導入細胞の製造方法
　本発明の遺伝子導入細胞の製造方法は、前記（１）に記載する本発明の核酸構築物または当該核酸構築物からの転写物を含むレトロウイルスベクターを細胞に導入する工程を包含することを特徴とする。本発明の遺伝子導入細胞の染色体には、本発明の核酸構築物の５’ＬＴＲと３’ＬＴＲの間に挟まれた領域に対応するＤＮＡが組み込まれている。本発明の一態様として、当該工程は体外で実施される。 (2) A method for producing a gene-introduced cell of the present invention The method for producing a gene-introduced cell of the present invention comprises transferring a retroviral vector containing the nucleic acid construct of the present invention described in (1) above or a transcript from the nucleic acid construct into a cell. characterized by comprising a step of introducing into 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 transfected cell of the present invention. In one aspect of the invention, the process is performed outside the body.
　本発明の方法は、哺乳動物、例えばヒト由来の細胞またはサル、マウス、ラット、ブタ、ウシ、イヌ等の非ヒト哺乳動物由来の細胞が使用できる。本発明の方法に使用される細胞に特に限定はなく、任意の細胞を使用することができる。例えば、血液（末梢血、臍帯血など）、骨髄などの体液、組織または器官より採取、単離、精製、誘導された細胞を使用することができる。末梢血単核細胞（ＰＢＭＣ）、免疫細胞［Ｔ細胞、樹状細胞、Ｂ細胞、造血幹細胞、マクロファージ、単球、ＮＫ細胞または血球系細胞（好中球、好塩基球）］、臍帯血単核球、線維芽細胞、前駆脂肪細胞、肝細胞、皮膚角化細胞、間葉系幹細胞、脂肪幹細胞、各種がん細胞株または神経幹細胞を使用することができる。本発明においては、特に免疫細胞、免疫細胞の前駆細胞（造血幹細胞、リンパ球前駆細胞等）またはこれらを含有する細胞集団の使用が好ましい。免疫系細胞の代表であるＴ細胞には、αβＴ細胞、γδＴ細胞、ＣＤ８陽性Ｔ細胞、ＣＤ４陽性Ｔ細胞、制御性Ｔ細胞、細胞傷害性Ｔ細胞、または腫瘍浸潤リンパ球が含まれる。Ｔ細胞およびＴ細胞の前駆細胞を含有する細胞集団には、ＰＢＭＣが含まれる。さらに、ＮＫ細胞、ＮＫＴ細胞やそれらの前駆細胞も本発明の方法の対象とすることができる。前記の細胞は生体より採取されたもの、それを拡大培養したもの、細胞株として樹立されたもの、多能性幹細胞から分化したもの等が例示されるが、これらに限定されるものではない。製造された遺伝子導入細胞または当該細胞より分化させた細胞を生体に移植することが望まれる場合には、その生体自身または同種の生体から採取された細胞より遺伝子導入細胞を製造することが好ましい。 The method 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. Cells used in the method of the present invention are not particularly limited, and any cells can be used. For example, cells collected, isolated, purified, and induced from blood (peripheral blood, umbilical cord blood, etc.), bodily fluids such as bone marrow, tissues or organs can be used. Peripheral blood mononuclear cells (PBMC), immune cells [T cells, dendritic cells, B cells, hematopoietic stem cells, macrophages, monocytes, NK cells or blood cells (neutrophils, basophils)], cord blood alone Nucleocytes, 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, it is particularly preferable to use immune cells, immune cell progenitor cells (hematopoietic stem cells, lymphocyte progenitor cells, etc.), or cell populations containing these cells. T cells, which are representative of immune system cells, include αβ T cells, γδ T cells, CD8 positive T cells, CD4 positive T cells, regulatory T cells, cytotoxic T cells, or tumor-infiltrating lymphocytes. Cell populations containing T cells and T cell progenitor cells include PBMCs. Furthermore, NK cells, NKT cells, and progenitor cells thereof can also be targeted by the methods of the present invention. Examples of the above-mentioned cells include, but are not limited to, those collected from living organisms, those obtained by expanding and culturing them, those established as cell lines, those differentiated from pluripotent stem cells, and the like. When it is desired to transplant the produced gene-introduced cells or cells differentiated from the cells into a living body, it is preferable to produce the gene-introduced cells from cells harvested from the living body itself or from the same kind of living body.
　本発明のレトロウイルスベクターを細胞に導入する工程で、導入効率を向上させる機能性物質を用いることもできる（例えば、国際公開第９５／２６２００号パンフレット、国際公開第００／０１８３６号パンフレット）。導入効率を向上させる物質としては、ウイルスベクターに結合する活性を有する物質、例えばフィブロネクチンまたはフィブロネクチンフラグメントなどの物質が挙げられる。好適には、ヘパリン結合部位を有するフィブロネクチンフラグメント、例えばレトロネクチン（ＲｅｔｒｏＮｅｃｔｉｎ、登録商標、ＣＨ－２９６、タカラバイオ社製）として市販されているフラグメントを用いることができる。市販されているレトロウイルス遺伝子導入の補助剤を使用してもよい。 In the step of introducing the retroviral vector of the present invention into cells, functional substances that improve the efficiency of introduction can be used (eg, International Publication No. 95/26200, International Publication No. 00/01836). Substances that improve transduction efficiency include substances that have activity to bind to viral vectors, such as fibronectin or fibronectin fragments. Preferably, a fibronectin fragment having a heparin-binding site, such as a fragment commercially available as RetroNectin (registered trademark, CH-296, manufactured by Takara Bio Inc.) can be used. Commercially available retroviral gene transfer aids may be used.
　本発明の好適な態様において、前記の機能性物質は各物質に適した方法で使用すればよく、例えばレトロネクチンの場合には適切な固相、例えば細胞培養に使用される容器（プレート、シャーレ、フラスコもしくはバッグ等）または担体（マイクロビーズ等）に固定化された状態で使用することができる。 In a preferred embodiment of the present invention, the functional substance may be used by a method suitable for each substance. flasks, bags, etc.) or immobilized on carriers (microbeads, etc.).
（３）本発明の細胞
　本発明の細胞は前記（２）の製造方法により製造された遺伝子導入細胞である。本発明の細胞は、外来性の所望の配列にコードされている遺伝子産物を発現する。したがって、本発明の遺伝子導入細胞は前記遺伝子産物に起因する新たな性質および／または機能を獲得している。 (3) Cell of the Present Invention The cell of the present invention is a gene-introduced cell produced by the production method of (2) above. The cells of the invention express the gene product encoded by the exogenous desired sequence. Thus, the transgenic cells of the present invention have acquired new properties and/or functions resulting from said gene product.
　本発明の一態様として、本発明の細胞は疾患の治療剤として使用することができる。該治療剤は、疾患の治療に有用な遺伝子産物を発現しうる本発明の細胞を有効成分として含み、さらに、適当な賦形剤を含んでいてもよい。該賦形剤としては薬学的に許容できるものであれば特に限定はないが、例えば、安定剤、緩衝剤、等張化剤などが挙げられる。本発明の細胞が投与される疾患としては、当該細胞に感受性を示す疾患であればよく、特に限定はないが、例えば、がん［血液がん（白血病）、固形腫瘍など］、炎症性疾患／自己免疫疾患（喘息、湿疹など）、肝炎や、ウイルス、細菌、真菌が原因となる感染性疾患（インフルエンザ、ＡＩＤＳ、結核、ＭＲＳＡ感染症、ＶＲＥ感染症、深在性真菌症が例示される。前記の疾患において減少もしくは消失が望まれる細胞が有している抗原、すなわち腫瘍抗原、ウイルス抗原、細菌抗原等を認識するＴＣＲやＣＡＲを発現する細胞がこれら疾患の治療のために投与される。また、本発明の細胞は、骨髄移植や放射線照射後の感染症予防、再発白血病の寛解を目的としたドナーリンパ球輸注などにも利用できる。本発明の細胞を活性成分として含む治療剤は、限定するものではないが、非経口投与、例えば、注射または注入により、皮内、筋肉内、皮下、腹腔内、鼻腔内、動脈内、静脈内、腫瘍内、または輸入リンパ管内などに投与することができる。 As one aspect of the present invention, the cells of the present invention can be used as therapeutic agents for diseases. The therapeutic agent contains, as an active ingredient, the cells of the present invention capable of expressing gene products useful for treating diseases, and may further contain suitable excipients. The excipient is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include stabilizers, buffers, tonicity agents and the like. Diseases to which the cells of the present invention are administered are not particularly limited as long as they are diseases that exhibit sensitivity to the cells, but examples include cancer [blood cancer (leukemia), solid tumors, etc.] and inflammatory diseases. /Autoimmune diseases (asthma, eczema, etc.), hepatitis, and infectious diseases caused by viruses, bacteria, and fungi (influenza, AIDS, tuberculosis, MRSA infection, VRE infection, and deep mycosis are exemplified) Cells expressing TCRs or CARs that recognize antigens possessed by cells whose reduction or elimination is desired in the above diseases, ie, tumor antigens, viral antigens, bacterial antigens, etc., are administered for the treatment of these diseases. The cells of the present invention can also be used for bone marrow transplantation, prevention of post-irradiation infections, transfusion of donor lymphocytes for the purpose of remission of recurrent leukemia, etc. A therapeutic agent comprising the cells of the present invention as an active ingredient is: , but not limited to parenteral administration, such as by injection or infusion, intradermally, intramuscularly, subcutaneously, intraperitoneally, intranasally, intraarterially, intravenously, intratumorally, or intraafferent lymphatics. be able to.
　以下に実施例を挙げて本発明をさらに具体的に説明するが、本発明は以下の実施例のみに限定されるものではない。
　また、本明細書に記載の操作のうち、基本的な操作については２００１年、コールド　スプリング　ハーバー　ラボラトリー発行、Ｔ．マニアティス（Ｔ．Ｍａｎｉａｔｉｓ）ら編集、モレキュラー　クローニング：ア　ラボラトリー　マニュアル第３版（Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ　３ｒｄ　ｅｄ．）に記載の方法によった。 EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited only to the following examples.
In addition, among the operations described in this specification, basic operations are described in 2001, Cold Spring Harbor Laboratory, T.W. The method described in T. Maniatis et al., Molecular Cloning: A Laboratory Manual 3rd ed.
実施例１　Ｇａｇ残存配列削減ウイルスベクタープラスミドの作製
　まず、ｐＭＳＣＶｎｅｏ（Ｃｌｏｎｔｅｃｈ社製）を鋳型として、ＰＣＲにより配列番号１に示すＭＳＣＶ　Ｕ３プロモーター配列のＤＮＡ断片を増幅した。つづいて、ｐＬＶＳＩＮ－ＩＲＥＳ－ＺｓＧｒｅｅｎ１Ｖｅｃｔｏｒを鋳型として、ＰＣＲにより配列番号２に示すＺｓＧｒｅｅｎ１配列のＤＮＡ断片を増幅した。これらのＤＮＡ断片をｐＬＶＳＩＮ－ＣＭＶ－Ｎｅｏ　（タカラバイオ社製）のＣｌａＩ－ＭｌｕＩ消化産物に挿入し、ｐＬＶＳＩＮ－ＭＳＣＶ－ＺｓＧｒｅｅｎ１プラスミドを作製した。 Example 1 Preparation of Gag Residual Sequence-Reduced Viral Vector Plasmid First, using pMSCVneo (manufactured by Clontech) as a template, a DNA fragment of the MSCV U3 promoter sequence shown in SEQ ID NO: 1 was amplified by PCR. Subsequently, using pLVSIN-IRES-ZsGreen1Vector as a template, a DNA fragment having the ZsGreen1 sequence shown in SEQ ID NO: 2 was amplified by PCR. These DNA fragments were inserted into the ClaI-MluI digested product of pLVSIN-CMV-Neo (manufactured by Takara Bio Inc.) to prepare the pLVSIN-MSCV-ZsGreen1 plasmid.
　次に、図１に示すように、ｐＬＶＳＩＮ－ＭＳＣＶ－ＺｓＧｒｅｅｎ１に含まれるＨＩＶ－１　Ｇａｇ配列由来の配列をそれぞれ１３３ｂｐ、１７７ｂｐ、２２２ｂｐ、２６７ｂｐ、１７８ｂｐ（４５＋１３３）削減したベクターＸ、ベクターＶ、ベクターＷ、ベクターＹ、ベクターＺを作製した。Ｇａｇ配列削減ベクターＸ、Ｖ、Ｗ、Ｙに含まれるＨＩＶ－１　Ｇａｇ配列由来の配列をそれぞれ配列番号３、４、５、６に記載する。 Next, as shown in FIG. 1, vector X, vector V, and vector W in which the sequence derived from the HIV-1 Gag sequence contained in pLVSIN-MSCV-ZsGreen1 was reduced by 133 bp, 177 bp, 222 bp, 267 bp, and 178 bp (45+133), respectively , vector Y, and vector Z were created. Sequences derived from the HIV-1 Gag sequences contained in Gag sequence-reduced vectors X, V, W, and Y are shown in SEQ ID NOs: 3, 4, 5, and 6, respectively.
実施例２　レトロウイルス溶液の作製
　実施例１で調製したｐＬＶＳＩＮ－ＭＳＣＶ－ＺｓＧｒｅｅｎ１およびベクターＸ、Ｖ、Ｗ，Ｙ、Ｚにより大腸菌ＪＭ１０９をそれぞれ形質転換した。これら形質転換体の保持するプラスミドＤＮＡをＮｕｃｌｅｏＳｐｉｎ（登録商標）　Ｐｌａｓｍｉｄ　Ｍｉｄｉ（マッハライナーゲル社製）を用いてそれぞれ精製し、トランスフェクション用ＤＮＡとして以下の操作に供した。調製したそれぞれのプラスミドと、パッケージングプラスミド（ＨＩＶ－１由来のＧａｇ、Ｐｏｌ、Ｔａｔ、Ｒｅｖのレンチウイルスタンパク質と、ＶＳＶ－Ｇエンベロープタンパク質が一過性に発現される）を２９３Ｔ細胞（ＡＴＣＣ　ＣＲＬ－１１２６８）に導入し、得られた細胞を培養して、ＶＳＶ－Ｇエンベロープを持つレンチウイルスを含有する上清液をそれぞれ調製した。上清液を０．４５μｍフィルター（Ｍｉｌｅｘ　ＨＶ、ミリポア社製）にてろ過し、それぞれウイルス溶液ＬＶＳＩＮ－ＭＳＣＶ－ＺｓＧｒｅｅｎ１、溶液Ｘ，溶液Ｙ、溶液Ｖ、溶液Ｗ、溶液Ｚとした。 Example 2 Preparation of Retrovirus Solution E. coli JM109 was transformed with pLVSIN-MSCV-ZsGreen1 prepared in Example 1 and vectors X, V, W, Y and Z, respectively. Plasmid DNAs carried by these transformants were purified using NucleoSpin (registered trademark) Plasmid Midi (manufactured by Mach Reiner Gel) and subjected to the following operations as DNAs for transfection. Each prepared plasmid and packaging plasmid (in which HIV-1-derived Gag, Pol, Tat, and Rev lentiviral proteins and VSV-G envelope protein are transiently expressed) were transferred to 293T cells (ATCC CRL- 11268), and the resulting cells were cultured to prepare supernatants containing lentiviruses having VSV-G envelopes. The supernatant was filtered through a 0.45 μm filter (Milex HV, manufactured by Millipore) to obtain virus solution LVSIN-MSCV-ZsGreen1, solution X, solution Y, solution V, solution W, and solution Z, respectively.
実施例３　Ｇａｇ配列削減レンチウイルスベクターの感染
　実施例２で作製したウイルス溶液を適宜希釈し、ヒトＴリンパ球性白血病由来細胞株ＳｕｐＴ１細胞（ＡＴＣＣ　ＣＲＬ－１９４２）に対し１回感染を行った。フローサイトメーターを使用し、ウイルス感染３、４日後の細胞のＺｓＧｒｅｅｎ１陽性細胞の割合と陽性細胞中の平均蛍光強度を測定し、下式に従ってウイルスタイターを算出した。 Example 3 Infection with Gag sequence-reduced lentiviral vector The virus solution prepared in Example 2 was appropriately diluted, and a human T-lymphocytic leukemia-derived cell line SupT1 cell (ATCC CRL-1942) was infected once. Using a flow cytometer, the percentage of ZsGreen1-positive cells in cells 3 and 4 days after virus infection and the average fluorescence intensity in the positive cells were measured, and the virus titer was calculated according to the following formula.
ウイルスタイター（ＩＦＵ／ｍＬ）＝感染細胞数×（陽性率％／１００）×ウイルス希釈倍率／感染時液量（ｍＬ） Virus titer (IFU/mL) = number of infected cells x (% positive rate/100) x virus dilution rate/liquid volume at time of infection (mL)
　ベクターＬＶＳＩＮ－ＭＳＣＶ－ＺｓＧｒｅｅｎ１を１００％とした時の平均蛍光強度を比較したものを図２に、ウイルスタイターを比較したものを図３に示す。いずれも、３回試験した値の平均値を算出している。図２に示すようにベクターＸ、Ｖ、Ｗ、ＹはベクターＬＶＳＩＮ－ＭＳＣＶ－ＺｓＧｒｅｅｎ１と同等の平均蛍光強度を示した。図３に示すようにベクターＷ，Ｙについては大幅なウイルスタイターの低下が見られた一方で、ベクターＸ，Ｖについては大きな低下は見られなかった。 　Figure 2 shows a comparison of the average fluorescence intensity when the vector LVSIN-MSCV-ZsGreen1 is 100%, and Figure 3 shows a comparison of the virus titers. All are calculating the average value of the value tested 3 times. As shown in FIG. 2, vectors X, V, W, and Y exhibited mean fluorescence intensities comparable to vector LVSIN-MSCV-ZsGreen1. As shown in FIG. 3, vectors W and Y showed a significant decrease in virus titer, while vectors X and V did not show a significant decrease.
実施例４　ハイブリッド型ＬＴＲおよびＷＰＲＥ２
　実施例１で調製したｐＬＶＳＩＮ－ＭＳＣＶ－ＺｓＧｒｅｅｎ１のＭＳＣＶ－Ｕ３プロモーター配列をヒトＥＦ１αプロモーター配列に置換したｐＬＶＳＩＮ－ＥＦ１α－ＺｓＧｒｅｅｎ１を作製した。さらに、前記２つのベクターの５’ＬＴＲ配列を、外来のプロモーター配列としてＣＭＶプロモーター配列を含むＬＴＲ配列（配列番号７）に置換してハイブリッド型のＬＴＲとし、残存するＧＡＧ配列を１７７ｂｐ削除して１８３ｂｐ（配列番号４）とした。さらに、ＷＰＲＥ配列を、当該配列に含まれるＸ　ｐｒｏｔｅｉｎの発現を抑制させるためにＸプロテインの開始コドンＡＴＧのＡを１位として６位と７位の間にフレームシフトを生じる挿入塩基を挿入したＷＰＲＥ２（配列番号８）に置換した。こうして、ｐＬＧＴ２－ＭＳＣＶ－ＺｓＧｒｅｅｎ１およびｐＬＧＴ２－ＥＦ１α－ＺｓＧｒｅｅｎ１をそれぞれ作製した。各ベクターの構造を図４に示す。 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 the 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 an exogenous promoter sequence to obtain a hybrid LTR, and the remaining GAG sequence was deleted by 177 bp to 183 bp. (SEQ ID NO: 4). Furthermore, the WPRE sequence is WPRE2 in which an insertion base that causes a frameshift between positions 6 and 7 with A of the start codon ATG of the X protein as 1 is inserted in order to suppress the expression of the X protein contained in the sequence. (SEQ ID NO: 8). Thus, pLGT2-MSCV-ZsGreen1 and pLGT2-EF1α-ZsGreen1 were constructed, respectively. The structure of each vector is shown in FIG.
　作製したプラスミドＤＮＡにより大腸菌ＨＳＴ０８をそれぞれ形質転換した。これら形質転換体の保持するプラスミドＤＮＡをＮｕｃｌｅｏＳｐｉｎ（登録商標）　Ｐｌａｓｍｉｄ　Ｍｉｄｉ（マッハライナーゲル社製）を用いてそれぞれ精製し、トランスフェクション用ＤＮＡとして以下の操作に供した。調製したプラスミドＤＮＡのそれぞれと、実施例２で使用したパッケージングプラスミドとを２９３Ｔ細胞にトランスフェクトし、ＶＳＶＧエンベロープを持つレンチウイルスを含有する４種の上清液を獲得した。なお、ｐＬＧＴ２－ＭＳＣＶ－ＺｓＧｒｅｅｎ１およびｐＬＧＴ２－ＥＦ１α－ＺｓＧｒｅｅｎ１には、レンチウイルスのＴＡＴタンパク質を発現するプラスミドを含まないパッケージングプラスミドを使用した。上清液を０．４５μｍフィルター（Ｍｉｌｅｘ　ＨＶ、ミリポア社製）にてろ過し、それぞれウイルス溶液を調製し、ＬＶＳＩＮ－ＭＳＣＶ、ＬＶＳＩＮ－ＥＦ１α、ＬＧＴ２－ＭＳＣＶおよびＬＧＴ２－ＥＦ１αとした。 Escherichia coli HST08 was transformed with each of the prepared plasmid DNAs. Plasmid DNA retained by these transformants was purified using NucleoSpin (registered trademark) Plasmid Midi (manufactured by Mach Reiner Gel), and subjected to the following operations as DNA for transfection. Each of the prepared plasmid DNAs and the packaging plasmid used in Example 2 were transfected into 293T cells to obtain four supernatants containing VSVG enveloped lentiviruses. For pLGT2-MSCV-ZsGreen1 and pLGT2-EF1α-ZsGreen1, a packaging plasmid containing no plasmid expressing the lentiviral TAT protein was used. The supernatant was filtered through a 0.45 μm filter (Milex HV, manufactured by Millipore) to prepare virus solutions, LVSIN-MSCV, LVSIN-EF1α, LGT2-MSCV and LGT2-EF1α.
　調製したウイルス溶液をＪ４５．０１細胞（ＡＴＣＣ　ＣＲＬ－１９９０）に種々の希釈率にて感染させた。ウイルス感染から４日後に細胞を回収し、フローサイトメーターを使用してＺｓＧｒｅｅｎ１遺伝子の発現細胞の割合を測定した。
　ＺｓＧｒｅｅｎ１の陽性率が１．０－２０．０％となる測定値を用いて、ウイルスタイターを算出した結果を図５に示す。図５に示す通り、いずれのレンチウイルスベクターも同等のウイルスタイターが得られた。 J45.01 cells (ATCC CRL-1990) were infected with the prepared virus solution at various dilutions. Cells were collected 4 days after virus infection, and the percentage of ZsGreen1 gene-expressing cells was measured using a flow cytometer.
FIG. 5 shows the results of calculating the virus titer using the measured values at which the ZsGreen1 positive rate was 1.0-20.0%. As shown in FIG. 5, equivalent viral titers were obtained for all lentiviral vectors.
　また、ｐＬＧＴ２－ＭＳＣＶ－ＺｓＧｒｅｅｎ１およびｐＬＧＴ２－ＥＦ１α－ＺｓＧｒｅｅｎ１のＷＰＲＥ配列に含まれるＸプロテインの発現を抑制させるためにＸプロテインの開始コドンＡＴＧのＡを１位として７位～９位を終止コドン（ＴＡＡ）に置換したＷＰＲＥ３（配列番号９）を含むプラスミドＤＮＡを作製した。前記と同様にウイルス溶液を調製し、Ｊ４５．０１細胞に感染させて、同等のウイルスタイターが得られることを確認した。 In addition, in order to suppress the expression of the X protein contained in the WPRE sequence of pLGT2-MSCV-ZsGreen1 and pLGT2-EF1α-ZsGreen1, the A of the start codon ATG of the X protein is the 1st position and the 7th to 9th positions are the stop codon (TAA A plasmid DNA was prepared containing WPRE3 (SEQ ID NO: 9) substituted with ). A virus solution was prepared in the same manner as described above and infected with J45.01 cells to confirm that equivalent virus titers were obtained.
　なお、ＷＰＲＥ配列に含まれるＸプロテインの発現について、野生型のＷＰＲＥ配列、ＷＰＲＥ２およびＷＰＲＥ３の３’末端に蛍光タンパク質ＡｃＧＦＰをコードする配列をそれぞれ連結し、ＸプロテインとＡｃＧＦＰの融合タンパク質の発現を確認した。蛍光強度の測定値から、ＷＰＲＥ２およびＷＥＰＲＥ３からＸプロテインが発現していないことが示され、ＷＰＲＥ２およびＷＥＰＲＥ３の高い安全性が確認された。 Regarding the expression of the X protein contained in the WPRE sequence, a sequence encoding the fluorescent protein AcGFP was ligated to the 3′ ends of the wild-type WPRE sequence, WPRE2 and WPRE3, respectively, and the expression of the fusion protein of the X protein and AcGFP was confirmed. bottom. Fluorescence intensity measurements showed that X protein was not expressed from WPRE2 and WEPRE3, confirming the high safety of WPRE2 and WEPRE3.
実施例５　ＣＡＲ遺伝子の発現
　実施例４で調製した各ベクタープラスミドＤＮＡのＺｓＧｒｅｅｎ１配列を、ｍｅｓｏｔｈｅｌｉｎ（ＭＳＬＮ）を特異的に認識するキメラ抗原受容体（ＣＡＲ）をコードする配列（ＭＳＬＮ－ＣＡＲ）に置換したプラスミドＤＮＡを調製した。それぞれ、ｐＬＶＳＩＮ－ＭＳＣＶ－ＭＳＬＮ－ＣＡＲ、ｐＬＶＳＩＮ－ＥＦ１α－ＭＳＬＮ－ＣＡＲ、ｐＬＧＴ２－ＭＳＣＶ－ＭＳＬＮ－ＣＡＲおよびｐＬＧＴ２－ＥＦ１α－ＭＳＬＮ－ＣＡＲとした。各ベクターの構造を図６に示す。 Example 5 Expression of CAR gene The ZsGreen1 sequence of each vector plasmid DNA prepared in Example 4 is replaced with a sequence (MSLN-CAR) encoding a chimeric antigen receptor (CAR) that specifically recognizes mesothelin (MSLN). A plasmid DNA was prepared. 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 FIG.
　作製したプラスミドにより、実施例４と同様の方法でそれぞれウイルス溶液を調製し、ＬＶＳＩＮ－ＭＳＣＶ、ＬＶＳＩＮ－ＥＦ１α、ＬＧＴ２－ＭＳＣＶおよびＬＧＴ２－ＥＦ１αとした。
　実施例４と同様の方法でウイルスタイターを測定した結果を図７に示す。図７に示す通り、通常のレンチウイルスベクターと比較して、ハイブリッド型のＬＴＲを含むプラスミドＤＮＡにより調製したレンチウイルスベクターは同等以上のウイルスタイターを示した。 Using the prepared plasmids, virus solutions were prepared in the same manner as in Example 4 to obtain LVSIN-MSCV, LVSIN-EF1α, LGT2-MSCV and LGT2-EF1α.
FIG. 7 shows the results of measuring the virus titer in the same manner as in Example 4. As shown in FIG. 7, the lentiviral vector prepared with the plasmid DNA containing the hybrid LTR showed equivalent or higher viral titer than the conventional lentiviral vector.
実施例６　ＣＡＲ遺伝子導入細胞の機能
　実施例５で調製した、ハイブリッド型のＬＴＲを含むプラスミドＤＮＡを使用し、ＭＳＬＮ－ＣＡＲ配列を含むウイルス溶液ＬＧＴ２－ＭＳＣＶおよびＬＧＴ２－ＥＦ１αを、ヒト末梢血より分離した末梢血単核球細胞（ＰＢＭＣ）に、種々の希釈率にてＲｅｔｒｏＮｅｃｔｉｎ（タカラバイオ社製）を用いて感染させた。ウイルス感染から７日後に細胞を回収し、ビオチン標識抗マウスＩｇＧ抗体（Ｊａｃｋｓｏｎ　ＩｍｍｕｎｏＲｅｓｅａｒｃｈ社製）とＦＩＴＣ標識抗Ｈｕｍａｎ　ＣＤ８　抗体（べクトンディッキンソン社製）を添加して細胞を染色し、フローサイトメーターを使用して抗ＭＳＬＮ－ＣＡＲ発現の陽性細胞の割合を測定した。結果を図８に示す。図８に示す通り、ＣＤ８陽性細胞において、抗ＭＳＬＮ－ＣＡＲ遺伝子が発現していることが確認された。 Example 6 Functions of CAR Gene-Introduced Cells Using the hybrid LTR-containing plasmid DNA prepared in Example 5, virus solutions LGT2-MSCV and LGT2-EF1α containing MSLN-CAR sequences were isolated from human peripheral blood. The obtained peripheral blood mononuclear cells (PBMC) were infected with RetroNectin (manufactured by Takara Bio Inc.) at various dilutions. Cells were collected 7 days after virus infection, biotin-labeled anti-mouse IgG antibody (manufactured by Jackson ImmunoResearch) and FITC-labeled anti-Human CD8 antibody (manufactured by Becton Dickinson) were added to stain the cells, and flow cytometry was performed. was used to measure the percentage of cells positive for anti-MSLN-CAR expression. The results are shown in FIG. As shown in FIG. 8, it was confirmed that the anti-MSLN-CAR gene was expressed in CD8-positive cells.
　また、ウイルス感染７日後に細胞を回収し、Ｒｅｃｏｍｂｉｎａｎｔ　ＤＮａｓｅＩ　（ＲＮＡｓｅ－ｆｒｅｅ）（タカラバイオ社製）を用いてウイルスに混入しているｐｌａｓｍｉｄ　ＤＮＡ等を分解した後、ＮｕｃｌｅｏＳｐｉｎ（登録商標）　Ｔｉｓｓｕｅ（マッハライナーゲル社製）を用いてゲノムＤＮＡを抽出し、ウイルスベクターの配列に特異的な遺伝子増幅用プライマーを用いてリアルタイムＰＣＲを行い、レンチウイルスベクターに由来するＤＮＡ量を定量した。さらにＩＦＮγ遺伝子の定量値に基づいてＤＮＡ量の補正を行うことにより、ゲノムに組み込まれたウイルスコピー数の測定を行った。結果を図９に示す。図９に示す通り、ＣＤ８陽性細胞において、ウイルス量に依存して抗ＭＳＬＮ－ＣＡＲ遺伝子がゲノムに組み込まれていることが確認された。 In addition, 7 days after virus infection, the cells were collected, and after decomposing plasmid DNA, etc. contaminating the virus using Recombinant DNase I (RNAse-free) (manufactured by Takara Bio Inc.), NucleoSpin (registered trademark) Tissue (Mach Reiner Gel Inc.) was used to extract genomic DNA, real-time PCR was performed using primers for gene amplification specific to the sequence of the viral vector, and the amount of DNA derived from the lentiviral vector was quantified. Furthermore, by correcting the amount of DNA based on the quantified value of the IFNγ gene, the virus copy number integrated into the genome was measured. The results are shown in FIG. As shown in FIG. 9, it was confirmed that the anti-MSLN-CAR gene was integrated into the genome in CD8-positive cells depending on the viral load.
　また、ウイルス溶液ＬＧＴ２－ＭＳＣＶおよびＬＧＴ２－ＥＦ１ａを感染させた細胞のうち、それぞれ１．９７コピーおよび０．８３コピーがゲノムに導入された細胞を回収した。ＭＳＬＮ陽性細胞であるＨｅＬａ細胞（ＡＴＣＣ　ＣＣＬ－２）およびＭＳＬＮ陰性細胞であるＫ５６２細胞（ＪＣＲＢ００１９）にＣａｌｃｅｉｎ　ＡＭ（ＰｒｏｍｏＣｅｌｌ社製）を取り込ませた後、前記ＣＡＲ発現細胞と１倍、３倍、１０倍の細胞数比で、４時間共培養した。共培養後、培養上清を回収し、蛍光強度を測定することで細胞障害性を算出した。結果を図１０に示す。図１０に示す通り、ＭＳＬＮ陽性細胞であるＨｅＬａ細胞に対するＣＡＲ発現細胞の細胞障害活性を確認した。 In addition, among the 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 collected. After incorporating Calcein AM (manufactured by PromoCell) into MSLN-positive HeLa cells (ATCC CCL-2) and MSLN-negative cells K562 cells (JCRB0019), the CAR-expressing cells were 1-fold, 3-fold, and 10-fold. The cells were co-cultured for 4 hours at a double cell number ratio. After co-cultivation, the culture supernatant was recovered, and the cytotoxicity was calculated by measuring the fluorescence intensity. The results are shown in FIG. As shown in FIG. 10, the cytotoxic activity of CAR-expressing cells against HeLa cells, which are MSLN-positive cells, was confirmed.
実施例７　ｓｉＲＮＡ発現細胞の機能
　国際公開第２０２１／０７０９５６号パンフレットに記載されるベクターＡおよびＤを用意した。ベクターＡは、５’から順に、５’ＬＴＲ、パッケージングシグナル配列、ｃＰＰＴ配列、ＲＲＥ配列、ＭＳＣＶ　Ｕ３プロモーター配列、ヒトＥＦ１α遺伝子由来のＳＤおよびＳＡ配列、２Ａペプチドによりポリシストロニックに連結したコドン変換型ＷＴ１特異的ＴＣＲα鎖とβ鎖遺伝子配列、ＷＰＲＥ配列を含む。ベクターＤは、ベクターＡのＷＰＲＥの下流に、ＴＣＲ遺伝子の発現を抑制する４種類のｓｉＲＮＡを生成する人工遺伝子を含む。なお、前記コドン変換型ＷＴ１特異的ＴＣＲα鎖とβ鎖遺伝子配列は、前記４種類のｓｉＲＮＡにより発現を抑制されないようにコドンが変換されている。前記ベクターＡおよびベクターＤを、ベクター１およびベクター２とした。 Example 7 Functions of siRNA-Expressing Cells Vectors A and D described in WO2021/070956 were provided. Vector A consists of, in order from 5′, 5′ LTR, packaging signal sequence, cPPT sequence, RRE sequence, MSCV U3 promoter sequence, human EF1α gene-derived SD and SA sequences, codon conversion linked polycistronic by 2A peptide. Includes type WT1-specific TCR α and β chain gene sequences, WPRE sequences. Vector D contains, downstream of WPRE of vector A, artificial genes that generate four types of siRNA that suppress TCR gene expression. The codon-converted WT1-specific TCR α-chain and β-chain gene sequences are codon-converted so that their expression is not suppressed by the four types of siRNA. Said vector A and vector D were referred to as vector 1 and vector 2, respectively.
　次に、ベクター１のＭＳＣＶ－Ｕ３プロモーター配列とヒトＥＦ１α遺伝子由来のＳＤおよびＳＡ配列をヒトＥＦ１αプロモーター配列に置換したベクター３を作製した。さらに、ベクター１の５’ＬＴＲ配列を外来のプロモーター配列としてＣＭＶプロモーター配列を含むＬＴＲ配列（配列番号７）に置換してハイブリッド型のＬＴＲとし、残存するＧＡＧ配列を１７７ｂｐ削除して１８３ｂｐ（配列番号４）とし、ｃＰＰＴ配列とＲＲＥ配列の順を入れ替え、ＷＰＲＥ配列をＷＰＲＥ２（配列番号８）に置換したベクター５を作製した。ベクター５のヒトＥＦ１α遺伝子由来のＳＤおよびＳＡ配列を削除したベクター４を作製した。また、ベクター４のＭＳＣＶ－Ｕ３プロモーター配列をヒトＥＦ１αプロモーター配列に置換したベクター６を作製した。各ベクターの構造を図１１に示す。 Next, Vector 3 was prepared by replacing the MSCV-U3 promoter sequence of Vector 1 and the SD and SA sequences derived from the human EF1α gene with the 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 obtain a hybrid LTR, and 177 bp of the remaining GAG sequence was deleted to 183 bp (SEQ ID NO: 4), the order of the cPPT sequence and the RRE sequence was reversed, and vector 5 was prepared by replacing the WPRE sequence with WPRE2 (SEQ ID NO: 8). Vector 4 was constructed by deleting the SD and SA sequences derived from the human EF1α gene of Vector 5. In addition, vector 6 was constructed by replacing the MSCV-U3 promoter sequence of vector 4 with the human EF1α promoter sequence. The structure of each vector is shown in FIG.
　作製したプラスミドにより、実施例４と同様の方法でそれぞれウイルス溶液を調製し、それぞれウイルス溶液１～６とした。ヒト末梢血より分離した末梢血単核球（ＰＢＭＣ）を、ＦＩＴＣ標識抗Ｈｕｍａｎ　ＣＤ８抗体（べクトンディッキンソン社製）にて染色し、ａｎｔｉ－ＦＩＴＣ　ｍｉｃｒｏｂｅａｄｓ（ミリテニバイオッテク社製）にてＣＤ８陽性細胞を分離した。ウイルス溶液１～６それぞれを２倍、６倍、１８倍、５４倍に希釈し、ＲｅｔｒｏＮｅｃｔｉｎ（タカラバイオ社製）を用いてこのＣＤ８陽性細胞に感染させた。ウイルス感染７日後に細胞を回収し、ＮｕｃｌｅｏＳｐｉｎ　ＲＮＡ　Ｐｌｕｓ（マッハライナーゲル社製）にて全ＲＮＡの抽出およびＤＮａｓｅＩ処理を行った。得られた全ＲＮＡを鋳型とし、ＰｒｉｍｅＳｃｒｉｐｔ　ＲＴ　ｒｅａｇｅｎｔ　Ｋｉｔ（Ｐｅｒｆｅｃｔ　Ｒｅａｌ　Ｔｉｍｅ）（タカラバイオ社製）を用いてｃＤＮＡ合成を行った。このｃＤＮＡを鋳型とし、ＴＢ　Ｇｒｅｅｎ　Ｐｒｅｍｉｘ　Ｅｘ　Ｔａｑ　ＩＩ（タカラバイオ社製）を用いたリアルタイムＰＣＲを行い、野生型ＴＣＲα鎖遺伝子、野生型ＴＣＲβ鎖遺伝子、コドン変換型ＴＣＲα鎖遺伝子、コドン変換型ＴＣＲβ鎖遺伝子の発現量を測定してその相対値を算出した。全ＲＮＡ量の補正はＧＡＰＤＨ遺伝子の発現量に基づいて行った。また、実施例６と同様の方法により、ゲノムに組み込まれたウイルスコピー数の測定を行った。 Using the prepared plasmid, each virus solution was prepared in the same manner as in Example 4 to obtain virus solutions 1 to 6, respectively. Peripheral blood mononuclear cells (PBMC) isolated from human peripheral blood were stained with FITC-labeled anti-Human CD8 antibody (manufactured by Becton Dickinson), and CD8 was detected with anti-FITC microbeads (manufactured by Militeni Biotech). Positive cells were isolated. Virus solutions 1 to 6 were diluted 2-fold, 6-fold, 18-fold and 54-fold, respectively, and retroNectin (manufactured by Takara Bio Inc.) was used to infect these CD8-positive cells. Cells were collected 7 days after virus infection, and total RNA was extracted and treated with DNase I using NucleoSpin RNA Plus (manufactured by Mach Reiner Gel). Using the obtained total RNA as a template, cDNA synthesis was performed using PrimeScript RT reagent Kit (Perfect Real Time) (manufactured by Takara Bio Inc.). Using this cDNA as a template, real-time PCR was performed using TB Green Premix Ex Taq II (manufactured by Takara Bio), wild-type TCRα chain gene, wild-type TCRβ chain gene, codon-converted TCRα chain gene, codon-converted TCRβ chain. The expression level of the gene was measured and the relative value was calculated. The total RNA amount was corrected based on the expression level of the GAPDH gene. In addition, by the same method as in Example 6, the virus copy number integrated into the genome was measured.
　ウイルスを感染させない陰性コントロール細胞の野生型ＴＣＲα鎖遺伝子、野生型ＴＣＲβ鎖遺伝子の発現量の相対値に基づき、各実験群での遺伝子発現の相対値の割合を算出することによって、野生型ＴＣＲ遺伝子の抑制効果を評価した。図１２に結果を示す。図１２中、縦軸はウイルスを感染させない陰性コントロール細胞の発現量を１００とした時の遺伝子の発現量の相対値を示す。横軸は、ウイルスコピー数を示す。図に示されるように、ベクター１は野生型ＴＣＲα鎖およびβ鎖遺伝子の発現を抑制せず、ベクター４、５、６は効率よく発現を抑制することを確認した。 Based on the relative expression levels of the wild-type TCR α chain gene and the wild-type TCR β chain gene in negative control cells not infected with the virus, the ratio of the relative values of gene expression in each experimental group was calculated to obtain the wild-type TCR gene. was evaluated for its inhibitory effect. The results are shown in FIG. In FIG. 12, the vertical axis indicates the relative value of the expression level of the gene when the expression level of the negative control cells not infected with the virus is set to 100. The horizontal axis indicates the virus copy number. As shown in the figure, vector 1 did not suppress the expression of wild-type TCR α chain and β chain genes, and vectors 4, 5 and 6 efficiently suppressed the expression.
　また、２倍希釈したベクター１のウイルス溶液を感染させた細胞のコドン変換型ＴＣＲα鎖、コドン変換型ＴＣＲβ鎖の遺伝子発現の相対値を基準とし、各実験群での遺伝子発現の相対値の割合を算出することによって、コドン変換型ＴＣＲ遺伝子の発現量を評価した。図１３に結果を示す。図１３中、縦軸は基準（２倍希釈したベクター１のウイルス溶液を感染させた細胞）の発現量を１００とした時の遺伝子の発現量の相対値を示す。横軸は、ウイルスコピー数を示す。図１３に示すように、ベクター３を導入した細胞は導入ウイルスのコピー数が低く、ベクター５、６を導入した細胞は、ベクター２、３を導入した細胞に比べて、同等のコドン変換型のヒト抗ＷＴ１　ＴＣＲ遺伝子を発現していることを確認した。 Also, relative values of gene expression of codon-converted TCRα chain and codon-converted TCRβ chain in cells infected with 2-fold diluted vector 1 virus solution are used as reference, and ratio of relative values of gene expression in each experimental group. The expression level of the codon-converted TCR gene was evaluated by calculating . The results are shown in FIG. In FIG. 13 , the vertical axis indicates the relative value of the expression level of the gene when the expression level of the reference (cells infected with the 2-fold diluted vector 1 virus solution) is set to 100. The horizontal axis indicates the virus copy number. As shown in FIG. 13, the cells into which vector 3 was introduced had a lower copy number of the introduced virus, and the cells into which vectors 5 and 6 were introduced had equivalent codon-conversion types than the cells into which vectors 2 and 3 were introduced. It was confirmed that the human anti-WT1 TCR gene was expressed.
　本発明により、効率よく所望の遺伝子が発現する核酸構築物、当該核酸構築物を細胞に導入するためのレトロウイルスベクター、当該ベクターを使用する遺伝子導入細胞の製造方法、当該ベクターが導入された細胞が提供される。これらの核酸構築物、レトロウイルスベクター、遺伝子導入細胞の製造方法および遺伝子導入細胞は、タンパク質の製造、細胞医療による疾患の治療およびそのための研究、試験に極めて有用である。 The present invention provides a nucleic acid construct that efficiently expresses a desired gene, a retroviral vector for introducing the nucleic acid construct into a cell, a method for producing a gene-introduced cell using the vector, and a cell into which the vector has been introduced. be done. These nucleic acid constructs, retroviral vectors, methods for producing gene-introduced cells, and gene-introduced cells are extremely useful for protein production, treatment of diseases by cell therapy, and studies and tests therefor.
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 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 (14)

  1. 　５’末端から順に、
    （ａ）外来のプロモーター配列を含むレトロウイルス由来の５’ＬＴＲ（Ｌｏｎｇ　Ｔｅｒｍｉｎａｌ　Ｒｅｐｅａｔ）配列、
    （ｂ）レトロウイルス由来のパッケージングシグナル配列（ψ）、
    （ｃ）１８３～２２７ｂｐの長さのｇａｇタンパク質をコードする核酸に由来する配列、
    （ｄ）所望の配列またはマルチクローニングサイト、
    （ｅ）レトロウイルス由来の３’ＬＴＲ配列、
    の各配列を含む、レトロウイルスベクターを製造するための核酸構築物。     From the 5' end,
    (a) a retrovirus-derived 5' LTR (Long Terminal Repeat) sequence containing an exogenous promoter sequence;
    (b) a packaging signal sequence (ψ) from a retrovirus,
    (c) a sequence derived from a nucleic acid encoding a gag protein between 183 and 227 bp in length;
    (d) a desired sequence or multiple cloning site;
    (e) a 3'LTR sequence from a retrovirus;
    A nucleic acid construct for producing a retroviral vector, comprising each sequence of
  2. 　さらに、Ｘプロテインをコードする核酸配列にフレームシフトを生じる配列またはＸプロテインの翻訳を中断する終止コドンが挿入された転写後調節配列（ＰＲＥ）を含む請求項１記載の核酸構築物。 The nucleic acid construct according to claim 1, further comprising a post-transcriptional regulatory sequence (PRE) inserted with a sequence that causes a frameshift in the nucleic acid sequence encoding the X protein or a stop codon that interrupts the translation of the X protein.
  3. 　ＰＲＥがウッドチャック肝炎ウイルス由来のＰＲＥ（ＷＰＲＥ）である請求項２記載の核酸構築物。 The nucleic acid construct according to claim 2, wherein the PRE is a woodchuck hepatitis virus-derived PRE (WPRE).
  4. 　外来のプロモーター配列がサイトメガロウイルス由来プロモーターである請求項１記載の核酸構築物。 The nucleic acid construct according to claim 1, wherein the exogenous promoter sequence is a cytomegalovirus-derived promoter.
  5. 　所望の配列が内部プロモーター配列を含む配列である請求項１記載の核酸構築物。 The nucleic acid construct of claim 1, wherein the desired sequence is a sequence containing an internal promoter sequence.
  6. 　所望の配列がＴ細胞受容体（ＴＣＲ）またはキメラ抗原受容体（ＣＡＲ）をコードする配列を含む請求項１記載の核酸構築物。 The nucleic acid construct of claim 1, wherein the desired sequence comprises a sequence encoding a T cell receptor (TCR) or chimeric antigen receptor (CAR).
  7. 　所望の配列が２Ａペプチドで連結されたＴＣＲα鎖およびＴＣＲβ鎖をコードする配列を含む請求項６記載の核酸構築物。 The nucleic acid construct of claim 6, wherein the desired sequence comprises sequences encoding TCRα and TCRβ chains linked by a 2A peptide.
  8. 　ＬＴＲ配列がレンチウイルス由来の配列である請求項１記載の核酸構築物。 The nucleic acid construct according to claim 1, wherein the LTR sequence is a lentivirus-derived sequence.
  9. 　３’ＬＴＲ配列が自己不活性化（ＳＩＮ）ＬＴＲ配列である請求項１記載の核酸構築物。 The nucleic acid construct of claim 1, wherein the 3'LTR sequence is a self-inactivating (SIN) LTR sequence.
  10. 　請求項１～９のいずれか１項記載の核酸構築物からの転写物を含むレトロウイルスベクター。 A retroviral vector comprising a transcript from the nucleic acid construct according to any one of claims 1-9.
  11. 　オンコレトロウイルスまたはレンチウイルス由来の５’ＬＴＲ、パッケージングシグナル配列および３’ＬＴＲを含む請求項１０記載のレトロウイルスベクター。 The retroviral vector according to claim 10, comprising a 5'LTR, a packaging signal sequence and a 3'LTR derived from an oncoretrovirus or a lentivirus.
  12. 　レトロウイルス粒子を生成する能力を有する細胞に請求項１～９のいずれか１項記載の核酸構築物を導入する工程を含む、レトロウイルスベクターの製造方法。 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. 　請求項１０または１１に記載のレトロウイルスベクターを細胞に導入する工程を含む遺伝子導入細胞の製造方法。 A method for producing a gene-introduced cell, comprising the step of introducing the retroviral vector according to claim 10 or 11 into a cell.
  14. 　細胞が免疫細胞、免疫細胞に分化しうる細胞またはそれらを含有する細胞集団である請求項１３記載の遺伝子導入細胞の製造方法。 The method for producing gene-introduced cells according to claim 13, wherein the cells are immune cells, cells capable of differentiating into immune cells, or cell populations containing them.
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