TW202321456A - Nucleic acid for producing retroviral vector - Google Patents

Abstract

According to the present invention, provided is a nucleic acid construct for producing a retroviral vector, comprising: in order from the 5' end to 3' end, (a) a 5' LTR (Long Terminal Repeat) sequence derived from a retrovirus and comprising a foreign promoter sequence, (b) a packaging signal sequence (ψ) derived from a retrovirus, (c) a sequence of 186-227 bp length that is derived from a nucleic acid encoding a gag protein, (d) a sequence of a gene of interest or a multiple cloning site, and (e) a 3' LTR sequence derived from a retrovirus.

Description

Nucleic acids for retroviral vector production

The present invention relates to a nucleic acid construct for producing a retroviral vector for expressing a desired gene in mammalian cells, a retroviral vector containing a transcript transcribed from the nucleic acid construct, and methods using the same Methods for producing genes into 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 using endophagy, electroporation, and gene guns. Viral vector technology has been widely used in the field of gene therapy from basic to clinical. For example, adenoviral vectors are suitable for instantaneous and large-scale expression of target genes in target cells, and retroviral vectors can stably integrate into the host chromosome. With long-term stable performance, this vector is expected to be used in the field of gene therapy for genetic diseases. In addition, this technology is also expected to be used in the field of genetically modified animals.

Retroviruses contain gag/pol genes encoding structural proteins of viral particles, proteases, reverse transcriptase, integrase and other precursor proteins, and env genes encoding envelope glycoproteins. The two ends of these genes are sandwiched by 5'LTR (Long Terminal Repeat), which is related to the transcription of the viral genome, reverse transcription from the viral genome, and the integration of the double-stranded DNA synthesized by reverse transcription into the host DNA. ) and 3'LTR. In order to maintain the infectivity of viral particles and prevent them from replicating themselves, the gene delivery system using retroviral particles is divided into one or two packaging constructs expressing gag/pol genes and env genes, and Transfer vector encoding RNA to be integrated into retroviral vector particles. The transfer vector contains LTR and packaging signal sequences targeting virions, knocks out most of the gag/pol gene and the env gene, and inserts, for example, the desired sequence. The RNA genome is transcribed from the promoter sequence of the 5'LTR of the transfer vector, and the transcript is packaged into virions under the guidance of the packaging signal sequence.

In order to further reduce the possibility of retrovirus self-replication, the industry has developed self-inactivating (SIN) retroviral vectors that delete the promoter sequence of the 3'LTR that functions as a promoter on the chromosome of the target cell. In the SIN-type vector, the transcriptional regulation of the desired gene is performed by an internal promoter located at the 3' end of the packaging signal sequence. SIN-type vectors are safer because the sequence closer to the 5' end than the internal promoter is not transcribed, so the possibility of generating self-replicating virus particles is extremely low. Carriers with improved safety have been reported so far (Non-Patent Document 1, Non-Patent Document 2).

The industry hopes to develop safer and more efficient vectors for expressing desired genes in mammalian cells. [Prior technical literature] [Non-patent literature]

[Non-patent document 1] "Molecular Therapy (Mol. Ther.)", Volume 25, No. 8, Pages 1790-1804 (2017) [Non-patent document 2] "J. Virology", Volume 73, No. 7, Pages 6171-6176 (1999)

[Problem to be solved by the invention]

The object of the present invention is to provide a retroviral vector for introducing and expressing exogenous genes into cells more safely and efficiently. [Technical means to solve problems]

In order to solve the above-mentioned problems, the present inventors made diligent efforts to discover a retroviral vector that can achieve both the expression and safety of the introduced gene, and thus completed the present invention.

That is, the summary of the present invention is as follows: [1] A nucleic acid construct for producing retroviral vectors, which contains the following sequences in order from the 5' end: (a) A 5'LTR (Long Terminal Repeat) sequence derived from a retrovirus containing an exogenous promoter sequence, (b) Packaging signal sequence (ψ) derived from retrovirus, (c) A sequence of 183 to 227 bp in length derived from a nucleic acid encoding gag protein, (d) Required sequences or multiple selection sites, (e) 3'LTR sequence derived from retrovirus. [2] The nucleic acid construct according to [1], further comprising a post-transcriptional regulatory sequence (PRE) inserted into the nucleic acid sequence encoding the X protein, with a sequence causing a frame shift or a stop codon that interrupts the translation of the X protein. [3] The nucleic acid construct as described in [2], wherein the PRE is PRE (WPRE) derived from woodchuck hepatitis virus. [4] The nucleic acid construct as described in [1], wherein the foreign promoter sequence is a promoter derived from cytomegalovirus. [5] The nucleic acid construct according to [1], wherein the required sequence is a sequence including an internal promoter sequence. [6] The nucleic acid construct according to [1], wherein the required sequence includes a sequence encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR). [7] The nucleic acid construct according to [6], wherein the required sequence includes a sequence encoding a TCRα chain and a TCRβ chain linked via a 2A peptide. [8] The nucleic acid construct as described in [1], wherein the LTR sequence is a sequence derived from a lentivirus. [9] The nucleic acid construct as described in [1], wherein the 3'LTR sequence is a self-inactivating (SIN) LTR sequence. [10] A retroviral vector comprising a transcript transcribed from the nucleic acid construct described in any one of [1] to [9]. [11] The retroviral vector as described in [10], which contains a 5'LTR, a packaging signal sequence and a 3'LTR derived from an oncogenic retrovirus (oncoretrovirus) or lentivirus. [12] A method for producing a retroviral vector, which includes the step of introducing the nucleic acid construct described in any one of [1] to [9] into a cell capable of producing retroviral particles. [13] A method for producing a gene into cells, which includes the following steps: introducing the retroviral vector described in [10] or [11] into the cell. [14] The method of producing a gene-introduced cell as described in [13], wherein the cells are immune cells, cells capable of differentiating into immune cells, or a cell cluster containing the same. [Effects of the invention]

According to the present invention, there are provided a highly safe nucleic acid construct for producing a retrovirus that efficiently expresses a desired gene, and a retroviral vector containing a transcription product transcribed from the nucleic acid construct, A method for producing a cell using the vector and a cell containing the nucleic acid construct. These nucleic acid constructs, retroviral vectors, and cells are extremely useful for protein production, cell therapy for disease treatment, and for research and testing thereof.

In this specification, "nucleic acid construct" means a nucleic acid containing a sequence not found in nature that is constructed in a manner that contains one or more functional units. Nucleic acids can be DNA and/or RNA, and can also include modified nucleic acids. Examples of shapes include cyclic, linear, double-stranded, single-stranded, extrachromosomal DNA molecules (plastids), slime bodies, and the like. Furthermore, in addition to the nucleic acid sequence encoding the gene, the nucleic acid construct may also contain a nucleic acid sequence including a control sequence (such as a promoter) linked in an operable manner (ie, in a manner capable of controlling transcription or translation), if necessary. The nucleic acid construct may contain other regulatory factors, functional sequences, linkers, etc. as needed.

In this specification, "LTR (long terminal repeat)" means a sequence containing 100 to 1000 base pairs that is repeated at both ends of proviral DNA such as retroviruses and retrotransposons. The LTR is composed of the regions of U3, R, and U5 that are related to the transcription of the viral genome, reverse transcription from the viral genome, and the integration of the double-stranded DNA synthesized by reverse transcription into the host DNA. The length of the IR sequence (inverted repeat region) located at the 5' end and 3' end of the provirus is 4 to 20 base pairs. U3 contains the transcription enhancer sequence and promoter sequence.

In this specification, the "packaging signal sequence" is also recorded as "psi sequence" or "ψ sequence", which refers to the necessary sequence to form the capsid of the retroviral RNA chain and package it into the virion during the formation of virions. Encoding cis-acting sequences. For example, it is the region from the 3' side of the main splice donor (SD) site to the gag start codon, or the region from the 3' side of the SD site including a part of the gag gene sequence.

In this specification, "required sequence (sequence of interest)" means an exogenous sequence that is expected to be temporarily or permanently inserted artificially (human operation) into a cell (such as the nuclear genome or cytoplasm of the cell). This The species sequence includes a gene sequence that is completely or partially heterologous to the cell to be introduced, and also includes a gene sequence with any mutations. Alternatively, it can also be identical to the endogenous gene naturally contained in the cell. Gene sequence. Here, "natural" means the natural state without human manipulation.

In this specification, "multiple cloning sites" means a cluster sequence including a plurality of restriction enzyme sites for cloning. The base sequence constituting the multiple selection cloning site or the type and number of restriction enzyme sites included are not particularly limited.

In this specification, "posttranscriptional regulatory element (PRE: posttranscriptional regulatory element)" means that it helps promote the polyadenylation of mRNA transcribed from genes in cells, promotes extranuclear transport of mRNA, or activates mRNA. Translated sequence. By inserting into the untranslated region of the desired gene contained in the nucleic acid construct of the present invention, the expression of the protein level of the desired gene is improved.

In this specification, "wild type" means the gene or gene product that is isolated from a naturally occurring source and is most frequently observed in a population. It can be a natural isolate or an artificial one. On the other hand, "mutant" means a gene or gene product whose sequence and/or functional properties have been changed compared to a wild-type gene or gene product. Mutant genes are produced due to natural mutations or sequence mutations through artificial modification of genes.

In this specification, "T cells" are also called T lymphocytes, which refer to cells derived from the thymus among the lymphocytes involved in immune responses. T cells include helper T cells, suppressor T cells, regulatory T cells, CTL, naïve T cells, memory T cells, αβ T cells expressing α chain and β chain TCR, and γδ T cells expressing γ chain and δ chain TCR. . "Cells capable of differentiating into T cells" are not particularly limited as long as they are cells that differentiate into T cells in vivo or under artificial stimulation. Examples include hematopoietic stem cells, pluripotent precursor cells, common lymphoid precursor cells, and T cells. Precursor cells, etc. Examples of "cell clusters containing T cells or cells capable of differentiating into T cells" include blood (peripheral blood, umbilical cord blood, etc.), bone marrow fluid, and peripheral blood collected, isolated, purified, and induced therefrom. Cell clusters of mononuclear cells (PBMC), blood cells, hematopoietic stem cells, umbilical cord blood monocytes, etc. In addition, various cell clusters derived from blood cells containing T cells can be used in the present invention. These cells can be activated in vivo or ex vivo by anti-CD3 antibodies or cytokines such as IL-2. These cells can be any collected from an organism or cultured in vitro. For example, a T cell cluster directly obtained from an organism or a T cell cluster that has been cryopreserved.

Hereinafter, the present invention will be described in detail. (1) Nucleic acid construct of the present invention When the nucleic acid construction system of the present invention is used to produce retroviral vectors, it contains the following sequences in order from the 5' end: (a) A 5'LTR (long terminal repeat) sequence derived from a retrovirus including an exogenous promoter sequence, (b) Packaging signal sequence (ψ) derived from retrovirus, (c) A sequence of 183 to 227 bp in length derived from a nucleic acid encoding gag protein, (d) Required sequences or multiple selection sites, (e) 3'LTR sequence derived from retrovirus. The nucleic acid construct of the present invention can be used to produce retroviral vectors. That is, by introducing the nucleic acid construct of the present invention into cells having the ability to produce retroviral particles, the retroviral vector of the present invention containing a transcript transcribed from the nucleic acid construct can be produced. The nucleic acid construct of the present invention can produce retroviral vectors with higher viral titer. This retroviral vector can introduce desired sequences into cells. When the retroviral vector of the present invention is introduced into cells containing the desired sequence, the expression efficiency of the desired sequence is relatively high.

Retroviruses are enveloped viruses with a positive-sense single-stranded RNA genome. As the main elements in the viral genome, there are 5'LTR sequence, SD sequence, packaging signal sequence, gag gene, pol gene, and SA sequence from its 5' end. , env gene, 3'LTR sequence. In the case of lentivirus described below, in addition to these elements, a plurality of accessory genes are also included. Among them, in a gene introduction system using a retroviral vector, the retroviral vector must have a 5'LTR sequence, a packaging signal sequence, and a 3'LTR sequence, and the nucleic acid of the present invention has all of these. Other gene products such as gag, pol, and env can be supplied by packaging cells carrying these genes. The required sequence is usually placed on the 3' side of the packaging signal sequence of the retroviral vector, and when it contains an SA sequence, it is placed on the 3' side of the packaging signal sequence and the SA sequence.

The nucleic acid construct of the present invention contains a sequence derived from a nucleic acid encoding gag protein with a length of 183 to 227 bp. The packaging signal sequence of retroviruses contains part of the gag gene sequence, so it cannot be completely removed. However, the sequence of the above length helps to efficiently produce the virus and express the required sequence. Furthermore, the risk of homologous recombination with the full-length gag gene used for producing viruses can be reduced. Therefore, the present invention can provide a method for producing a highly safe viral vector. For example, the sequence derived from the nucleic acid encoding gag protein is preferably 183 bp or 227 bp in length, and may exemplify the sequence described in SEQ ID NO: 4 or 3, or one or more sequences may be substituted, deleted, inserted, or added to the sequence. , for example, a base sequence of 1 to 9 bases.

The nucleic acid construct of the present invention contains (a) a 5'LTR sequence including an exogenous promoter sequence, (e) a 3'LTR sequence and (b) a packaging signal sequence as long as they are derived from retroviruses and can produce Any RNA having these sequences can be used as the sequence of a retrovirus having a genome. Retroviruses include subtypes of oncogenic retroviruses and lentiviruses, and sequences derived from viruses of either subtype may also be used in the present invention. These sequences can be sequences derived from the same virus, or sequences derived from different viruses can be combined to the extent that they can form viral particles by combining with appropriate packaging cells or can be integrated into the genome of the introduced cell.

The LTR sequence and packaging signal sequence used in the present invention can be derived from, for example, Moloney murine leukemia virus (MMLV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), which are oncogenic retroviruses. Sequences of mandibular proliferative sarcoma virus (MPSV) and spleen focus-forming virus (SFFV). Viral vectors derived from oncogenic retroviruses can introduce genes with high efficiency, but cells need to be actively dividing during vector introduction. These oncogenic retroviral vectors are described in many documents [for example, U.S. Patent No. 5,219,740, U.S. Patent No. 6,207,453, U.S. Patent No. 5,219,740, "BioTechniques" Vol. 7, No. 980~ Page 990 (1989), "Human Gene Therapy" Volume 1, Pages 5-14 (1990), "Virology" Volume 180, Pages 849-852 (1991), "U.S. National Proceedings of the Academy of Sciences (Proc. Natl. Acad. Sci. USA)", Volume 90, Pages 8033~8037 (1993), and "Contemporary Perspectives on Genetics and Development (Cur. Opin. Genet. Develop.)", Volume 3, No. Pages 102-109 (1993)].

In addition, the LTR sequence and packaging signal sequence used in the present invention can be derived from, for example, lentiviruses such as human immunodeficiency virus (HIV-1, HIV-2), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). ), Equine Infectious Anemia Virus (EIAV), Caprine Arthritis Encephalitis Virus (CAEV) sequences. Lentiviral vectors can introduce genes into the nuclear genome regardless of whether the cell into which the gene is introduced is in mitosis. Lentiviral vectors have also been described in many documents [for example, "J. Virology", Vol. 72, pp. 8463-8471 (1998)]. Other groups of retroviruses, such as foamy viruses (eg, foamy viruses), are also able to efficiently transduce into non-dividing cells.

Starting from the 5' end, LTR is divided into three regions: U3, R, and U5 according to function. The U3 region has enhancer/promoter activity and transcribes the R region of the 5'LTR sequence of the viral genome to the R region of the 3'LTR sequence under the action of RNA polymerase II of the host cell. The 5'LTR sequence used in the present invention contains an enhancer/promoter that is exogenous to the virus from which the LTR is derived. As one aspect of the present invention, the U3 region of the 5'LTR is replaced with an exogenous enhancer/promoter. Furthermore, an LTR sequence obtained by replacing the U3 region of the 3'LTR sequence with an enhancer/promoter derived from a virus other than the source virus of the LTR sequence can also be used in the present invention. Replacement exogenous enhancers/promoters may use sequences derived from viruses or mammalian sources, and constitutive, inducible or tissue-specific enhancers/promoters may be used. For example, human cytomegalovirus (HCMV) immediate early (immediate early), Moloney mouse sarcoma virus (MMSV), mouse stem cell virus (MSCV), Rous sarcoma virus (RSV), spleen focus-forming virus (SFFV) can be used ) and other virus-derived enhancers/promoters, or mammalian-derived enhancers/promoters of β-actin, hemoglobin, elastase, albumin, α-fetoprotein and insulin genes. Here, the term enhancer/promoter refers to a sequence including an enhancer region and/or a promoter region. In general, the enhancer region and promoter region are sometimes collectively referred to as promoters. In order to distinguish it from the enhancer region, the promoter region is sometimes called a core promoter. Either enhancer region and/or promoter region may be used in the present invention. As one aspect of the present invention, the 5'LTR sequence including the exogenous promoter sequence includes the sequence described in SEQ ID NO: 7, or one or several, for example, 1 to 9, are substituted, deleted, inserted, or added to the sequence. The base sequence of bases.

The 3'LTR sequence used in the present invention can be a sequence in which a mutation is introduced into the U3 region to delete the enhancer/promoter activity. Thereby, when a viral vector containing a transcript transcribed from the nucleic acid construct infects a cell, the viral genome is integrated into the provirus formed by the cell chromosome, and transcription from the R region is inhibited. In this way, retroviral vectors with mutations in the U3 region of the 3'LTR sequence are called self-inactivating (SIN) vectors. The introduction of mutations into the 3'LTR sequence is carried out by substitution or deletion of bases. In SIN-type vectors, since transcription from the R region of the LTR is inhibited in the provirus, the promoter sequence used to express the desired sequence needs to be configured separately from the LTR. Furthermore, when a plurality of required sequences are expressed, a promoter sequence different from the LTR is also configured. Sometimes, the promoter sequence that exists between the 5'LTR and the 3'LTR is called an internal promoter sequence. The internal promoter sequence may be a promoter sequence derived from a virus or a mammalian gene. When a promoter sequence derived from a virus is used, a sequence derived from the same virus as the source virus of the 5'LTR, 3'LTR or packaging signal sequence or a sequence derived from a different virus may be used. 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 mouse stem cell virus (MSCV), can be used. In addition, as the internal promoter sequence, the sequence exemplified in the previous paragraph as an exogenous promoter replacing the U3 region of the 5'LTR sequence can be used. As the virus-derived promoter sequence, in addition to the above, sequences such as SV40 promoter and CMV promoter can also be used. Furthermore, 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. Waiting sequence. Furthermore, the internal promoter can be placed upstream of (d) (i.e., the 5' side). It can be placed between (a) and (d), and it is suitable to be placed between (b) and (d).

The nucleic acid construct of the invention may comprise SD sequences and/or SA sequences. These sequences may use SD sequences and/or SA sequences that are exogenous to the LTR, or SD sequences and/or SA sequences that are exogenous to the internal promoter sequence. In addition, the SD sequence and the SA sequence may be sequences from different sources. For example, 16S RNA derived from simian virus (SV) 40, HCMV immediate early RNA, SD sequence and SA sequence of human hEF1α gene can be used [Proceedings of the National Academy of Sciences of the United States of America, Vol. 95, No. 1, No. 219-223 Page (1998)]. In addition, SD sequences or SA sequences in which mutations are introduced into common sequences to enhance or inhibit splicing activity can also be used in the nucleic acid construct of the present invention.

The (d) desired sequence contained in the nucleic acid construct of the present invention is a sequence intended to be expressed in cells introduced into the produced vector. The sequence includes, for example, a sequence encoding a protein, a sequence encoding a tRNA or a miRNA, or RNA that functions within the cell. Alternatively, a sequence (multiple selection cloning site) composed of a plurality of restriction enzyme recognition sequences for linking the desired sequence can be produced and arranged as the sequence (d), and then the multiple selection cloning site can be used. to insert the desired sequence. The nucleic acid constructs of the present invention also include such nucleic acid constructs having multiple selection sites in place of the desired sequences.

The above-mentioned desired sequences may be those aimed at preventing or treating diseases. Examples include: sequences used to inhibit the transcription or expression of harmful gene products in organisms (such as sequences encoding siRNA), sequences encoding proteins used to supplement proteins that are missing or have lost their functions in organisms, and sequences that can change or strengthen cells. The sequence of functions it has, etc. The present invention provides gene therapy, which involves introducing into an organism cells containing exogenous sequences via the nucleic acid construct of the present invention. For example, the use of sequences encoding IL-2 receptor gamma chain (X-linked severe combined immunodeficiency), sequences encoding β-globin (β-thalassemia), and sequences encoding adenosine deaminase (ADA) (ADA Deficiency), gene therapy for sequences encoding coagulation factors (hemophilia), and sequences encoding receptors that recognize antigens (cancer or viral infections).

As one aspect of the present invention, the desired sequence contained in the nucleic acid construct of the present invention is a sequence encoding an oligomer protein. Oligomeric proteins include structural proteins, enzymes, transcription factors, receptors, and antibodies. Furthermore, in the present invention, the oligomer protein may also be a cell surface protein (membrane protein), and the required sequence is preferably a sequence encoding an antigen recognition receptor, such as a T cell receptor (TCR), as exemplified in the examples.

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). The structure of a representative CAR is composed of a single chain antibody (single chain variable fragment: scFv (single chain variable fragment)) that recognizes the surface antigen of target cells to be eliminated from the organism, such as tumor cells, a transmembrane region, and a T Components of the intracellular domain of cell activation. As the intracellular region, it is preferable to use the intracellular region of the TCR complex CD3ζ. CARs with this structure are called first-generation CARs. Genes for the single chain antibody portion may, for example, be isolated from fusion tumors that produce monoclonal antibodies that recognize the target antigen. T cells expressing CAR directly recognize the surface antigens of target cells regardless of the expression of major histocompatibility antigen class I on tumor cells, and activate T cells at the same time, thereby effectively killing target cells.

In order to enhance the T cell activation ability of the first generation CAR, a second generation CAR was developed that is linked to the intracellular domain of costimulatory molecules of T cells. As costimulatory molecules for T cells, it is appropriate to use the intracellular region of CD28, tumor necrosis factor (TNF) receptor superfamily CD137 (4-1BB) or CD134 (OX40). As a further improvement, third-generation CARs have been developed in tandem with the intracellular regions of these costimulatory molecules, and a variety of CAR molecules targeting various tumor antigens have been reported. The nucleic acid construct of the present invention may also include a sequence encoding any CAR as a desired sequence.

Nucleic acid constructs of the invention may also contain post-transcriptional regulatory sequences (PREs) that help enhance the expression of the desired sequence. PREs are located in introns of transcripts transcribed from nucleic acid constructs and can be removed by splicing during the life cycle of retroviruses. Examples of PREs that cannot be removed by shearing include post-transcriptional processing elements of herpes simplex virus, post-transcriptional regulatory sequences of hepatitis B virus (HPRE) and woodchuck hepatitis virus (WPRE). The PRE sequence contains the coding region for protein X, which is implicated in oncogenicity. The nucleic acid construct of the present invention may also use a mutant sequence derived from a PRE sequence that inhibits the expression of protein X. For example, a PRE sequence can be used to insert a sequence that causes a frame shift or a stop codon that interrupts the translation of the X protein into the nucleic acid sequence encoding the X protein. As one aspect of the present invention, A in the start codon ATG of protein base PRE sequence. Alternatively, a PRE sequence in which positions 7 to 9 are replaced with a stop codon (eg, TAA) can be used. In the present invention, WPRE is preferred. For WPRE, refer to US Patent No. 6,136,597 or US Patent No. 7,419,829. As one aspect of the present invention, WPRE2 (SEQ ID NO: 8) or WPRE3 (SEQ ID NO: 9) can be used, or one or several bases, for example, 1 to 9 bases, can be substituted, deleted, inserted, or added to the sequence. base sequence. As one aspect of the present invention, a nucleic acid construct in which a PRE sequence is arranged between (d) a desired sequence or multiple selection site and (e) a 3'LTR sequence derived from a retrovirus is exemplified.

The nucleic acid construct of the invention may comprise a Rev response element (RRE) sequence and/or a central polypurine tract (cPPT) sequence. Examples of RREs include, but are not limited to, RREs located at positions 7622 to 8459 of the HIV NL4-3 genome (GenBank accession number: AF003887), RREs derived from other strains of HIV or other retroviruses. In addition, cPPT is a sequence of approximately 15 bases located in the approximate center of the lentiviral genome, and this sequence functions as a primer binding site for synthesizing positive-strand DNA during the synthesis of double-stranded DNA from lentiviral genomic RNA. When reverse-transcribing the RNA genome of lentivirus, it functions together with the central termination sequence (CTS) to form a three-stranded structure called a DNA flap. Furthermore, in the nucleic acid construct of the present invention, the arrangement of the two sequences is not particularly limited. cPPT and RRE may be arranged in sequence from the 5' end, or RRE and cPPT may be arranged in sequence.

The present invention includes a method for producing retroviral vectors (particles), which includes the step of introducing the nucleic acid construct of the present invention into cells capable of producing retroviral particles. In order to stably exert an effect in cells, the nucleic acid construct can be introduced into cells using appropriate vectors such as plasmid vectors, viral vectors other than retroviruses, or transposon vectors. Furthermore, genome editing technology can be used to integrate into the chromosomal DNA of cells. In addition, the nucleic acid construct can be directly introduced into cells. When the nucleic acid construct is introduced directly into cells or using a plastid carrier, methods using carriers such as liposomes and ligand-polylysine, calcium phosphate method, electropenetration method, particle gun method, etc. can be used. The above-mentioned viral vector is not particularly limited, and known viral vectors commonly used in gene introduction methods can be used, such as adenovirus vectors, adeno-associated virus vectors, monkey virus vectors, vaccinia virus vectors, measles virus vectors, or Sendai virus vectors.

As cells having the ability to produce retroviral particles, for example, known packaging cells can be used. Appropriate packaging cells can be selected based on the LTR sequence and packaging signal sequence of the nucleic acid construct, and the above nucleic acid construct can be introduced to produce retrovirus-producing cells and prepare retrovirus vectors (particles). In addition, the nucleic acid construct of the present invention and the nucleic acid encoding the components required to produce retroviral particles (gag gene, pol gene, env gene or other accessory genes) can be introduced simultaneously or randomly into cells with higher transfection efficiency ( 293 cells or 293T cells, etc.), the cells are used as retrovirus-producing cells and cultured in a suitable medium to produce retroviral particles. The retroviral particles produced contain transcripts transcribed from the nucleic acid construct of the invention. This retroviral particle is included in the present invention as a retroviral vector for introducing a desired sequence into a cell. Examples of packaging cells include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), and the like. Kits containing packaging cells or other cells, and plasmids for producing retroviruses (called packaging plasmids) for producing retroviruses are widely commercially available from various companies, and these commercial products can be used in the method of the present invention.

In the present invention, pseudotypes (pseudotypes) can be produced by using packaging cells expressing an envelope protein derived from a heterologous virus that is different from the virus from which the retroviral vector genome is derived, or a packaging plasmid containing a sequence encoding the above-mentioned envelope protein. pseudotyped) retrovirus. For example, a packaging plasmid encoding an envelope derived from MMLV, gibbon leukemia virus (GaLV), vesicular stomatitis virus (VSV), feline endogenous virus, or a protein capable of functioning as an envelope may be used. Furthermore, packaging cells introduced with enzyme genes involved in sugar chain synthesis can be used to produce retroviral vectors having sugar chain-modified proteins on their surfaces.

After the retrovirus-producing cells produced by the above operations are cultured, the cell culture is centrifuged and the supernatant is recovered, and appropriate filtration operations are used to remove impurities to obtain retroviral particles. The crude retroviral particles can be directly contacted with cells for gene introduction, or higher purity retroviral particles can be prepared through known purification operations and used for gene introduction operations.

The present invention provides a composition, which uses the retroviral vector of the present invention as an active ingredient and also contains pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are well-known in the industry, including, for example, phosphate buffered saline (for example, 0.01 M phosphate, 0.138 M NaCl, 0.0027 M KCl, pH 7.4), containing hydrochloride, hydrobromide, and phosphate. , aqueous solutions of inorganic acid salts such as sulfate, physiological saline, solutions such as ethylene glycol or ethanol, and organic acid salts such as acetate, propionate, malonate, and benzoate. Additives such as wetting agents or emulsifiers, and pH buffers can also be used. As pharmaceutically acceptable excipients, those described in "Remington's Pharmaceutical Sciences" (Mack Pub. Co., N.J. 1991) (which becomes a part of this specification by clearly stating the source) can be appropriately used. The composition can take a known form suitable for parenteral administration, such as injection or infusion. Furthermore, preparation auxiliaries such as suspending agents, preservatives, stabilizers and/or dispersants, and preservatives for extending the effective shelf life can be used. The compositions may also be in a dry form for reconstitution with a suitable sterile liquid prior to use.

(2) Method for producing the gene-introduced cells of the present invention The method for producing genes into cells of the present invention is characterized by including the following steps: introducing into cells a retroviral vector containing the nucleic acid construct of the present invention described in the above (1) or a transcript transcribed from the nucleic acid construct. . The DNA corresponding to the region sandwiched between the 5'LTR and the 3'LTR of the nucleic acid construct of the present invention is integrated into the chromosome of the cell introduced into the gene of the present invention. As one aspect of the present invention, this step is carried out in vitro.

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. The 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.), body 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) can be used )], umbilical cord blood mononuclear cells, fibroblasts, precursor adipocytes, liver cells, skin keratinocytes, mesenchymal stem cells, adipose stem cells, various cancer cell lines or neural stem cells. In the present invention, it is particularly preferable to use immune cells, immune cell precursor cells (hematopoietic stem cells, lymphocyte precursor cells, etc.) or cell clusters containing the same. T cells as representative 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 precursor cells include PBMCs. Furthermore, NK cells, NKT cells or these precursor cells can also be used as targets of the method of the present invention. Examples of the above-mentioned cells include, but are not limited to, those collected from living organisms, those expanded and cultured, those constructed 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 an organism, it is preferable to produce the gene-introduced cells from cells collected from the organism itself or a similar organism.

In the step of introducing the retroviral vector of the present invention into cells, functional substances that improve the efficiency of introduction can also be used (for example, International Publication No. 95/26200 Pamphlet, International Publication No. 00/01836 Pamphlet). Examples of substances that improve the efficiency of introduction include substances that have the activity of binding to viral vectors, such as fibronectin or fibronectin fragments. Fibronectin fragments having a heparin-binding site, such as commercially available fragments of RetroNectin (registered trademark, CH-296, manufactured by TAKARA BIO Co., Ltd.), can be preferably used. Commercially available retroviral gene transfer auxiliaries can also be used.

In a preferred aspect of the present invention, the above-mentioned functional substances can be used by a method suitable for each substance. For example, in the case of RetroNectin, they can be fixed to a suitable solid phase such as a container (culture plate, petri dish) used for cell culture. , flask or bag, etc.) or carrier (microbeads, etc.).

(3) Cells of the present invention The cell system of the present invention is a gene-transfected cell produced by the production method of (2) above. The cells of the present invention express gene products encoded by exogenous desired sequences. Therefore, the cells introduced with the genes of the present invention acquire new properties and/or functions derived from the above gene products.

As one aspect of the present invention, the cells of the present invention can be used as a therapeutic agent for diseases. The therapeutic agent contains cells of the present invention capable of expressing gene products useful for treating diseases as active ingredients, and may further contain appropriate excipients. The excipient is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include stabilizers, buffers, isotonic agents, and the like. The diseases to which the cells of the present invention are administered are not particularly limited as long as they show sensitivity to the cells. Examples include: cancer [blood cancer (leukemia), solid tumors, etc.], inflammatory diseases/autologous diseases Immune diseases (asthma, eczema, etc.), hepatitis, or infectious diseases caused by viruses, bacteria, and fungi (influenza, AIDS, tuberculosis, MRSA infection, VRE infection, deep mycosis). In order to treat these diseases, cells expressing TCR or CAR are administered. The TCR or CAR recognizes the antigens contained in the cells that are expected to be reduced or eliminated in the above diseases, that is, tumor antigens, viral antigens, bacterial antigens, etc. In addition, the cells of the present invention can also be used for donor lymphocyte infusion, etc., to prevent infections after bone marrow transplantation or radiation irradiation, and to alleviate the recurrence of leukemia. The therapeutic agent containing the cells of the present invention as an active ingredient is not limited, and can be administered intradermally, intramuscularly, subcutaneously, intraperitoneally, intranasally, intraarterially, or intravenously by parenteral administration such as injection or infusion. , within the tumor, or within the afferent lymphatic vessels, etc. [Example]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited only to the following examples. In addition, the basic operations described in this manual are in accordance with the 3rd edition of "Molecular Cloning: A Laboratory Manual" (2001, published by Cold Spring Harbor Laboratory, T. The method described in Maniatis et al. (edited by).

Example 1 Preparation of viral vector plasmid with deletion of Gag residual sequence First, a DNA fragment of the MSCV U3 promoter sequence shown in SEQ ID NO: 1 was amplified by PCR using pMSCVneo (manufactured by Clontech) as a template. Then, using the pLVSIN-IRES-ZsGreen1 vector as a template, PCR was used to amplify the DNA fragment of the ZsGreen1 sequence shown in SEQ ID NO: 2. These DNA fragments were inserted into the ClaI-MluI digestion product of pLVSIN-CMV-Neo (manufactured by TAKARA BIO) to prepare pLVSIN-MSCV-ZsGreen1 plasmid.

Next, as shown in Figure 1, a vector was produced in which the sequences derived from the HIV-1 Gag sequence contained in pLVSIN-MSCV-ZsGreen1 were deleted by 133 bp, 177 bp, 222 bp, 267 bp, and 178 bp (45+133) respectively. X, vector V, vector W, vector Y, vector Z. The sequences derived from the HIV-1 Gag sequence contained in the Gag sequence deletion vectors X, V, W, and Y are recorded as sequence numbers 3, 4, 5, and 6 respectively.

Example 2 Preparation of retrovirus solution Coliform JM109 was transformed using pLVSIN-MSCV-ZsGreen1 and vectors X, V, W, Y, and Z prepared in Example 1 respectively. The plasmid DNA carried by the transformants was purified using NucleoSpin (registered trademark) Plasmid Midi (manufactured by MACHEREY-NAGEL) and used as transfection DNA in the following operation. Each prepared plasmid and packaging plasmid (which transiently expresses lentiviral proteins derived from Gag, Pol, Tat, Rev and VSV-G envelope protein of HIV-1) were introduced into 293T cells (ATCC CRL-11268), The obtained cells were cultured, and supernatants containing lentiviruses with VSV-G envelope were prepared. The supernatant was filtered using a 0.45 μm filter (Milex HV, manufactured by Millipore) to prepare virus solutions LVSIN-MSCV-ZsGreen1, solution X, solution Y, solution V, solution W, and solution Z respectively.

Example 3 Infection of Gag sequence-deleted lentiviral vector The virus solution prepared in Example 2 was appropriately diluted, and the human T lymphocytic leukemia-derived cell line SupT1 cells (ATCC CRL-1942) were infected once. Use a flow cytometer to measure the ratio of ZsGreen1-positive cells and the average fluorescence intensity in positive cells 3 or 4 days after virus infection, and calculate the virus titer according to the following formula.

Virus titer (IFU/mL) = number of infected cells × (positive rate %/100) × virus dilution rate/fluid volume during infection (mL)

The comparison results of the average fluorescence intensity when the vector LVSIN-MSCV-ZsGreen1 is set to 100% are shown in Figure 2, and the comparison results of the virus titers are shown in Figure 3. Calculate the average of the values obtained from the three tests. As shown in Figure 2, vectors X, V, W, and Y showed the same average fluorescence intensity as the vector LVSIN-MSCV-ZsGreen1. As shown in Figure 3, a significant decrease in the virus titer of vectors W and Y was observed, while on the other hand, no significant decrease was observed in vectors X and V.

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 above two vectors were replaced with the LTR sequence (SEQ ID NO: 7) containing the CMV promoter sequence as an exogenous promoter sequence to create a hybrid LTR, and the remaining GAG sequence was deleted by 177 bp. Becomes 183 bp (sequence number 4). Furthermore, the WPRE sequence was replaced with WPRE2 (SEQ ID NO: 8). In this WPRE2, the A of the start codon ATG of protein X was set to position 1 and an insertion base was inserted between positions 6 and 7 to cause a frame shift. To inhibit the expression of the X protein contained in this sequence. In this way, pLGT2-MSCV-ZsGreen1 and pLGT2-EF1α-ZsGreen1 were produced respectively. The structure of each carrier is shown in Figure 4.

The prepared plasmid DNA was used to transform Escherichia coli HST08. The plasmid DNA carried by the transformants was purified using NucleoSpin (registered trademark) Plasmid Midi (manufactured by MACHEREY-NAGEL) and used as transfection DNA in the following operation. Each prepared plasmid DNA and the packaging plasmid used in Example 2 were transfected into 293T cells, and four types of supernatants containing lentiviruses with VSVG envelope were obtained. In addition, for pLGT2-MSCV-ZsGreen1 and pLGT2-EF1α-ZsGreen1, a packaging plasmid other than the plasmid expressing the lentiviral TAT protein was used. The supernatant was filtered using a 0.45 μm filter (Milex HV, manufactured by Millipore) to prepare each virus solution as LVSIN-MSCV, LVSIN-EF1α, LGT2-MSCV, and LGT2-EF1α.

The prepared virus solutions were used to infect J45.01 cells (ATCC CRL-1990) at various dilution rates. Cells were recovered 4 days after virus infection, and the rate of ZsGreen1 gene-expressing cells was measured using flow cytometry. The virus titer was calculated using the measured value when the positive rate of ZsGreen1 reached 1.0 to 20.0%, and the results are shown in Figure 5. As shown in Figure 5, each lentiviral vector obtained the same viral titer.

Furthermore, a plasmid DNA containing WPRE3 (SEQ ID NO: 9) was prepared, in which the A of the start codon ATG of protein Inhibits the expression of X protein contained in the WPRE sequence of pLGT2-MSCV-ZsGreen1 and pLGT2-EF1α-ZsGreen1. Prepare the virus solution in the same manner as above and infect J45.01 cells to confirm that the same virus titer is obtained.

Furthermore, regarding the expression of the X protein contained in the WPRE sequence, sequences encoding the fluorescent protein AcGFP were connected to the 3' ends of the wild-type WPRE sequence, WPRE2, and WPRE3 to confirm the expression of the fusion protein of the X protein and AcGFP. According to the measured value of fluorescence intensity, it is shown that WPRE2 and WEPRE3 do not express X protein, confirming that WPRE2 and WEPRE3 have higher safety.

Example 5 Expression of CAR gene Plasmid DNA was produced by replacing the ZsGreen1 sequence of each vector plasmid DNA prepared in Example 4 with a sequence encoding a chimeric antigen receptor (CAR) that specifically recognizes mesothelin (MSLN) (MSLN-CAR). As pLVSIN-MSCV-MSLN-CAR, pLVSIN-EF1α-MSLN-CAR, pLGT2-MSCV-MSLN-CAR and pLGT2-EF1α-MSLN-CAR respectively. The structure of each carrier is shown in Figure 6 .

Using the prepared plasmid, each virus solution was prepared as LVSIN-MSCV, LVSIN-EF1α, LGT2-MSCV and LGT2-EF1α in the same manner as in Example 4. The virus titer was measured in the same manner as in Example 4, and the results are shown in Figure 7 . As shown in Figure 7, compared with ordinary lentiviral vectors, lentiviral vectors prepared using plasmid DNA containing hybrid LTR showed equal or higher viral titers.

Example 6 Function of CAR gene introduced into cells RetroNectin (manufactured by TAKARA BIO) was used to infect human beings at various dilution rates with the virus solutions LGT2-MSCV and LGT2-EF1α prepared in Example 5 using plasmid DNA containing hybrid LTR and containing the MSLN-CAR sequence. Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood. The cells were recovered 7 days after virus infection, and 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 the expression of anti-MSLN- was measured using a flow cytometer. Ratio of CAR-positive cells. The results are shown in Figure 8 . As shown in Figure 8, it was confirmed that CD8-positive cells expressed the anti-MSLN-CAR gene.

In addition, cells were recovered 7 days after virus infection, and Recombinant DNase I (RNAse-free) (manufactured by TAKARA BIO Co., Ltd.) was used to decompose plastid DNA mixed in the virus, etc., and then NucleoSpin (registered trademark) Tissue (manufactured by MACHEREY-NAGEL Co., Ltd.) was used. Genomic DNA is extracted, and real-time PCR is performed using gene amplification primers specific to the viral vector sequence to quantify the amount of DNA derived from the lentiviral vector. Furthermore, based on the quantitative value of the IFNγ gene, the DNA amount was corrected to determine the viral copy number integrated into the genome. The results are shown in Figure 9. As shown in Figure 9, it was confirmed that the anti-MSLN-CAR gene was integrated into the genome of CD8-positive cells in a virus-amount-dependent manner.

Furthermore, among the cells infected by the virus solutions LGT2-MSCV and LGT2-EF1a, cells whose genomes had introduced virus copy numbers of 1.97 and 0.83 were recovered, respectively. After integrating calcein acetate methyl ester (Calcein AM) (manufactured by PromoCell) into HeLa cells (ATCC CCL-2), which are MSLN-positive cells, and K562 cells (JCRB0019), which are MSLN-negative cells, they were pressed with the above-mentioned CAR-expressing cells. Co-culture was carried out for 4 hours at cell number ratios of 1x, 3x, and 10x. After co-culture, the culture supernatant was recovered and the fluorescence intensity was measured to calculate the cytotoxic activity. The results are shown in Figure 10. As shown in Figure 10, the cytotoxic activity of the CAR-expressing cells against HeLa cells, which are MSLN-positive cells, was confirmed.

Example 7 siRNA expresses the function of cells Prepare carriers A and D described in International Publication No. 2021/070956 Pamphlet. Vector A contains 5'LTR, packaging signal sequence, cPPT sequence, RRE sequence, MSCV U3 promoter sequence, SD and SA sequences derived from human EF1α gene in sequence from 5', via 2A peptide polycistronic ), the codon-switchable WT1-specific TCR α chain and β chain gene sequences and WPRE sequences are linked to each other. Vector D contains artificial genes downstream of the WPRE of vector A that generate four types of siRNA that inhibit the expression of TCR genes. Furthermore, the codon-switched WT1-specific TCR α chain and β chain gene sequences are codon-switched in such a way that their expression is not inhibited by the above four siRNAs. Let the above-mentioned carrier A and carrier D be carrier 1 and carrier 2.

Next, vector 3 was prepared by replacing the MSCV-U3 promoter sequence of vector 1 with the SD and SA sequences derived from the human EF1α gene, replacing the human EF1α promoter sequence. Furthermore, vector 5 was produced by replacing the 5'LTR sequence of vector 1 with an LTR sequence (SEQ ID NO: 7) containing a CMV promoter sequence as an exogenous promoter sequence to create a hybrid LTR, and the remaining GAG sequence was 177 bp was deleted to become 183 bp (SEQ ID NO: 4), the order of the cPPT sequence and the RRE sequence was reversed, and the WPRE sequence was replaced by WPRE2 (SEQ ID NO: 8). Vector 4 was produced by deleting the SD and SA sequences derived from the human EF1α gene of vector 5. Furthermore, vector 6 was prepared by replacing the MSCV-U3 promoter sequence of vector 4 with the human EF1α promoter sequence. The structure of each carrier is shown in Figure 11.

Using the prepared plasmid, each virus solution was prepared in the same manner as in Example 4, and was designated as virus solutions 1 to 6 respectively. Peripheral blood mononuclear cells (PBMC) isolated from human peripheral blood were stained using FITC-labeled anti-human CD8 antibodies (manufactured by Becton Dickinson), and CD8-positive cells were isolated using anti-FITC microbeads (manufactured by Miltenyi Biotec). Virus solutions 1 to 6 were diluted to 2 times, 6 times, 18 times, and 54 times respectively, and the CD8-positive cells were infected using RetroNectin (manufactured by TAKARA BIO). Seven days after virus infection, the cells were recovered, and total RNA was extracted using NucleoSpin RNA Plus (manufactured by MACHEREY-NAGEL) and treated with DNase I. Using the obtained total RNA as a template, cDNA was synthesized using PrimeScript RT reagent Kit (Perfect Real Time) (manufactured by TAKARA BIO). Using this cDNA as a template, real-time PCR was performed using TB Green Premix Ex Taq II (manufactured by TAKARA BIO) to measure the wild-type TCRα chain gene, wild-type TCRβ chain gene, codon-switched TCRα chain gene, and codon-switched TCRβ chain. The expression amount of the gene is calculated to calculate its relative value. Based on the expression level of GAPDH gene, the total RNA amount was corrected. Furthermore, by the same method as Example 6, the copy number of the virus integrated into the genome was measured.

Based on the relative values of the expression amounts of the wild-type TCRα chain gene and the wild-type TCRβ chain gene in negative control cells that were not infected with the virus, the ratio of the relative value of gene expression in each experimental group was calculated to evaluate the inhibitory effect of the wild-type TCR gene. . The results are shown in Figure 12. In FIG. 12 , the vertical axis represents the relative value of the gene expression level when the expression level of negative control cells not infected with the virus is set to 100. The horizontal axis represents virus copy number. As shown in the figure, it can be confirmed that vector 1 does not inhibit the expression of wild-type TCR α chain and β chain genes, and vectors 4, 5, and 6 effectively inhibit the expression.

Furthermore, the relative values of gene expression of codon-switched TCRα chain and codon-switched TCRβ chain in cells infected with a 2-fold diluted virus solution of vector 1 were used as a benchmark to calculate the relative values of gene expression in each experimental group. ratio to evaluate the expression of codon-switching TCR genes. The results are shown in Figure 13. In FIG. 13 , the vertical axis represents the relative value of the expression level of the gene when the expression level of the reference (cells infected with a virus solution diluted twice as much as vector 1) is 100. The horizontal axis represents virus copy number. As shown in Figure 13, it was confirmed that the copy number of the introduced virus in the cells introduced with vector 3 was lower. Compared with the cells introduced with vectors 2 and 3, the cells introduced with vectors 5 and 6 showed equivalent codons. Switching human anti-WT1 TCR gene. [Industrial availability]

According to the present invention, there are provided a nucleic acid construct that expresses a desired gene efficiently, a retroviral vector for introducing the nucleic acid construct into a cell, a method for producing a gene into a cell using the vector, and a device into which the vector is introduced. cells. These nucleic acid constructs, retroviral vectors, methods of producing genes into cells, and gene-transfection cells are extremely useful for protein production, cell therapy for disease treatment, and for research and testing thereof. [Sequence listing non-keyword text]

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 [sequence list]

Figure 1 is a diagram showing the structure of the nucleic acid construct produced in the Examples. Figure 2 is a graph showing the relative value of the fluorescence intensity of ZsGreen1 protein expressed in cells infected with retroviral vectors produced using each nucleic acid construct. Figure 3 is a graph showing the relative values of virus titers of retrovirus solutions produced using each nucleic acid construct. Fig. 4 is a diagram showing the structure of the nucleic acid construct produced in the Examples. Figure 5 is a graph showing the virus titer of retrovirus solutions produced using each nucleic acid construct. Fig. 6 is a diagram showing the structure of the nucleic acid construct produced in the Example. Figure 7 is a graph showing the virus titer of a retrovirus solution produced using each nucleic acid construct. Figure 8 is a graph showing the ratio of cells expressing positive CAR protein among cells infected with retroviral vectors produced using each nucleic acid construct. Figure 9 is a graph showing the number of viral copies integrated into the genome of cells infected with retroviral vectors produced using each nucleic acid construct. Figure 10 is a graph showing the cytotoxic activity of CAR-expressing cells infected with retroviral vectors produced using each nucleic acid construct. Fig. 11 is a diagram showing the structure of the nucleic acid construct produced in the Example. Figure 12 is a graph showing the RNA expression amount of wild-type TCR protein expressed in cells infected with retroviral vectors produced using each nucleic acid construct. Figure 13 is a graph showing the amount of RNA expression of the codon-switchable TCR protein expressed in cells infected with retroviral vectors produced using each nucleic acid construct.

TW202321456A_111133847_SEQL.xml

Claims (14)

A nucleic acid construct used to produce retroviral vectors, which contains the following sequences in sequence from the 5' end: (a) A 5'LTR (long terminal repeat) sequence derived from a retrovirus including an exogenous promoter sequence, (b) Packaging signal sequence (ψ) derived from retrovirus, (c) A sequence of 183 to 227 bp in length derived from a nucleic acid encoding gag protein, (d) Required sequences or multiple selection sites, (e) 3'LTR sequence derived from retrovirus. Such as the nucleic acid construct of claim 1, which further includes a post-transcriptional regulatory sequence (PRE) inserted into the nucleic acid sequence encoding the X protein to cause a frame shift or a stop codon that interrupts the translation of the X protein. The nucleic acid construct of claim 2, wherein PRE is PRE (WPRE) derived from woodchuck hepatitis virus. The nucleic acid construct of claim 1, wherein the foreign promoter sequence is a promoter derived from cytomegalovirus. Such as the nucleic acid construct of claim 1, wherein the required sequence is a sequence including an internal promoter sequence. Such as the nucleic acid construct of claim 1, wherein the required sequence includes a sequence encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR). Such as the nucleic acid construct of claim 6, wherein the required sequence includes a sequence encoding a TCRα chain and a TCRβ chain linked by a 2A peptide. Such as the nucleic acid construct of claim 1, wherein the LTR sequence is a sequence derived from a lentivirus. Such as the nucleic acid construct of claim 1, wherein the 3'LTR sequence is a self-inactivating (SIN) LTR sequence. A retroviral vector comprising a transcript transcribed from the nucleic acid construct of any one of claims 1 to 9. For example, the retroviral vector of claim 10 includes a 5'LTR, a packaging signal sequence and a 3'LTR derived from an oncogenic retrovirus or lentivirus. A method for manufacturing a retroviral vector, which includes the following steps: introducing the nucleic acid construct according to any one of claims 1 to 9 into cells capable of producing retroviral particles. A method of manufacturing a gene into a cell, which includes the following steps: introducing the retroviral vector of claim 10 or 11 into the cell. For example, 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 clusters containing the same.

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