Optimization of 3'LTR upstream sequence in lentiviral vector

Abstract

Provided is a nucleic acid construct that contains a nucleic acid encoding a lentiviral vector, wherein the lentiviral vector includes, in order from the 5'side thereof, a 5'LTR, a packaging signal, and a 3'LTR, and has a sequence composed of 10-65 nucleotides derived from a nef gene adjacent to the 5'side of the 3'LTR.

Description

Optimization of the 3'LTR upstream sequence in lentiviral vectors

[0001] The present invention relates to a nucleic acid construct for producing lentiviral vector particles used for introducing a desired gene into mammalian cells, a method for producing lentiviral vector particles using the nucleic acid construct, and a method for gene introduction using the viral vector particles.

[0002] Methods for introducing genes into eukaryotes include the use of viral vectors and techniques for introducing naked DNA via endocytosis, electroporation, and particle guns. Viral vectors are a widely used technique in gene therapy, from basic to clinical applications. For example, adenovirus vectors are suitable for transiently overexpressing target genes in target cells. Retroviral vectors, on the other hand, are promising vectors for gene therapy for genetic diseases and other conditions because they enable long-term stable expression of introduced genes through stable integration into host chromosomes. They are also a promising technique in the field of transgenic animal creation.

[0003] Lentivirus belongs to the Retroviridae family of viruses, but because it has several accessory proteins and regulatory proteins, it can infect non-dividing cells and introduce a nucleotide sequence derived from the viral genome into the host genome. Lentivirus is very different from other retroviruses (such as oncoretroviruses) in this regard, and in recent years, development has advanced as a vector for gene transfer. Lentivirus has a gag / pol gene encoding precursor proteins such as the structural proteins of the virus particle, protease, reverse transcriptase, and integrase, and an env gene encoding an envelope glycoprotein. At both ends of the viral genome, there are 5' LTR (Long Terminal Repeat) and 3' LTR involved in the transcription of the viral genome, reverse transcription using the viral genome as a template, and the integration of double-stranded DNA synthesized through reverse transcription into the host DNA. In a gene transfer system using lentivirus particles, the transfer vector encoding RNA incorporated into the viral vector particles does not contain genes encoding the structural proteins and regulatory proteins of the virus. When the transfer vector is introduced into cells endowed with the ability to express these proteins, virus particles in which the RNA genome transcribed from the transfer vector is packaged are generated in this cell.

[0004] A concern when using a lentivirus vector is the emergence of a replicating lentivirus. In order to further reduce the possibility that the lentivirus vector acquires the ability to self-replicate, a self-inactivating (SIN) type lentivirus vector with a deleted promoter of the 3' LTR that functions as a promoter on the chromosome of the target cell has been developed. In addition, a lentivirus vector with reduced sequences derived from wild-type lentivirus has been created (Patent Document 1, Non-Patent Document 1).

[0005] International Publication Pamphlet WO2023 / 038055

[0006] Molecular Therapy (Mol. Ther.), Vol. 25, No. 8, pp. 1790 - 1804 (2017)

[0007] The object of the present invention is to provide nucleic acid constructs for producing lentiviral vector particles that can introduce exogenous genes into cells more safely and efficiently.

[0008] As a result of our diligent efforts to solve the above problems, the inventors have discovered that by using a nucleic acid construct containing nucleic acid encoding a lentiviral vector in which the sequence derived from the nef gene has been reduced, lentiviral vector particles with a higher titer than conventional ones can be produced, thus completing the present invention.

[0009] In other words, the present invention relates to: [1] a nucleic acid construct comprising nucleic acid encoding a lentiviral vector, wherein the lentiviral vector comprises a 5'LTR, a packaging signal, and a 3'LTR in that order from its 5' side, and has a sequence of 10 to 65 bases derived from the nef gene adjacent to the 5' side of the 3'LTR; [2] the nucleic acid construct according to [1], wherein the sequence derived from the nef gene is the sequence shown in Sequence ID No. 4; [3] the nucleic acid construct according to [1] or [2], wherein the U3 region or a part of the 3'LTR is deleted; [4] the nucleic acid construct according to any one of [1] to [3], wherein a polyadenylation signal is present on the 3' side of the 3'LTR; [5] the nucleic acid construct according to any one of [1] to [4], wherein the U3 region or a part of the 5'LTR is replaced with an exogenous promoter; [6] the nucleic acid construct according to any one of [1] to [5], wherein an internal promoter is included between the packaging signal and the 3'LTR; [7] The present invention relates to a nucleic acid construct according to [6], comprising a multi-cloning site or an exogenous gene functionally linked to the internal promoter between an internal promoter and the 3'LTR; [8] a nucleic acid construct according to [6], comprising a PRE between an internal promoter and the 3'LTR; [9] a nucleic acid construct according to [7], comprising a PRE between a multi-cloning site or an exogenous gene functionally linked to the internal promoter and the 3'LTR;

[10] lentivirus-producing cells into which any of the nucleic acid constructs according to [1] to [9] have been introduced;

[11] a method for producing lentivirus vector particles comprising the steps of culturing the lentivirus-producing cells according to

[10] and collecting lentivirus vector particles from the cell culture obtained in the above step; and a method for introducing a gene into target cells comprising the steps of producing lentivirus vector particles by the method according to

[11] and infecting target cells with the lentivirus vector particles obtained in the above step.

[0010] The present invention provides a nucleic acid construct that has a viral genome in which specific sequences derived from wild-type lentiviruses have been reduced, and that can produce high-titer lentiviral vector particles; a method for producing lentiviral vector particles using the nucleic acid construct; and a method for introducing genes into target cells using the lentiviral vector particles. The present invention is extremely useful in the fields of biotechnology, medicine, and agriculture / livestock farming.

[0011] This figure shows the sequence derived from the nef gene held by the lentiviral vector. This figure shows the average fluorescence intensity (relative value) of the fluorescent protein in lentiviral vector-infected cells. This figure shows the titer of lentiviral vector particles in cell culture supernatant. This figure shows the structure of the lentiviral vector plasmid. This figure shows the structure of the lentiviral vector plasmid. This figure shows the structure of the lentiviral vector plasmid. This figure shows the structure of the lentiviral vector plasmid. This figure shows the titer of lentiviral vector particles in cell culture supernatant. This figure shows the structure of the lentiviral vector. This figure shows the titer of lentiviral vector particles in cell culture supernatant. This figure shows the titer of lentiviral vector particles in cell culture supernatant. This figure shows the fluorescent protein positivity rate in lentiviral vector-infected cells. This figure shows the structure of the lentiviral vector. This figure shows the titer of lentiviral vector particles in cell culture supernatant. This figure shows the CAR expression positivity rate in lentiviral vector-infected cells.

[0012] A "lentiviral vector" refers to a vector system for gene transfer created using a lentivirus as a base. In this specification, RNA or DNA containing the viral genome sequence of a lentiviral vector may be referred to as a lentiviral vector. Viral particles in which the RNA genome of a lentiviral vector is packaged are referred to as lentiviral vector particles.

[0013] In this specification, “nucleic acid construct” means a nucleic acid containing a sequence constructed to include a combination of multiple functional units not found in nature. Nucleic acids may be DNA and / or RNA, and may also include modified nucleic acids. Examples of shapes include circular, linear, double-stranded, single-stranded, extrachromosomal DNA molecules (plasmids), and cosmids. In addition to nucleic acid sequences encoding proteins or functional RNA, nucleic acid constructs may also include regulatory sequences (promoters and other regulatory elements) and linkers that are operably linked to other sequences (i.e., capable of controlling transcription and translation).

[0014] In this specification, "LTR (Long Terminal Repeat)" refers to the sequences located at both ends of the proviral DNA of lentiviruses. LTRs are involved in the transcription of the viral genome, reverse transcription from the viral genome, and the integration of the double-stranded DNA synthesized through reverse transcription into the host DNA, and their structure consists of the U3, R, and U5 regions. The U3 region contains the transcription enhancer and promoter.

[0015] In this specification, "packaging signal" is also referred to as "psi sequence" or "ψ sequence," and refers to a cis-active sequence necessary for capsid formation of the lentiviral RNA strand and packaging into the viral particle during viral particle formation. In lentiviruses, it is the region containing a portion of the gag gene at the 3' end of the primer binding site (PBS).

[0016] Nef (negative factor) is a protein of approximately 27 kDa, and the nef gene encoding this protein is located in the 3' region of the HIV-1 genome, from the 3' portion of the env gene to the 5' portion of the 3' LTR. The Nef protein was once thought to be a factor that suppresses viral replication, but it has since been revealed to have the function of reducing the expression of HLA class I molecules and CD4 receptors in host cells, and is involved in the enhancement of viral replication and increased infectivity within cells.

[0017] Eukaryotic mRNA has a poly(A) tail at its 3' end. This poly(A) tail is not encoded on the template DNA, but is added during the processing of precursor RNA, which is produced by transcription from DNA, into mature RNA. The DNA region that determines the poly(A) addition site, corresponding to a position approximately 10 to 30 bases from the 5' end of the mature RNA, is called the polyadenylation signal (poly(A) addition signal).

[0018] In this specification, "multicloning site" refers to a collection of multiple nucleotide sequences recognized by restriction enzymes. There are no particular restrictions on the types and number of nucleotide sequences that make up a multicloning site, or the restriction enzyme recognition sites contained therein.

[0019] In this specification, "posttranscriptional regulatory element (PRE)" refers to a sequence that contributes to promoting polyadenylation (addition of poly-A to mRNA transcribed from a gene within a cell), promoting the export of mRNA from the nucleus, or activating mRNA translation. By inserting it into the untranslated region of a desired gene contained in the nucleic acid construct of the present invention, the expression level of the desired gene is improved. The Rev response element (RRE) is the region to which the Rev protein, an accessory gene product of the lentiviral, binds, and is necessary for the transport of the viral genome from the nucleus to the cytoplasm. The central polyprint lacte (cPPT) is a sequence of approximately 15 bases located near the center of the lentiviral genome, and is a sequence that acts as a primer binding site for positive-strand DNA synthesis during the process of double-strand DNA synthesis from lentiviral genomic RNA. When the lentiviral RNA genome is reverse transcribed, it functions together with the central termination sequence (CTS) to form a triple-stranded structure called a DNA flap.

[0020] In this specification, the “exogenous” element in a lentiviral vector means an element that the lentivirus does not inherently possess (derived from another virus or a different type of cell).

[0021] In this specification, “gene of interest” means an exogenous gene that is desired to be introduced into a cell, either temporarily or permanently, through artificial manipulation. Such genes include genes that are entirely or partially heterologous to the cell into which they are introduced, and genes with any arbitrary mutations. They may also be genes with sequences identical to endogenous genes naturally present in the cell. “Naturally” here means in a natural state without artificial manipulation.

[0022] In this specification, “wild-type” means a gene or gene product isolated from a naturally occurring source that is most frequently observed in a population. This can be isolated from nature or artificially created. On the other hand, “mutant” refers to a gene or gene product in which the sequence and / or functional characteristics have been altered compared to the wild-type gene or gene product. Mutant genes are produced by spontaneous mutation or by artificially modifying the gene to alter its sequence.

[0023] The present invention will be described in detail below. (1) Nucleic acid construct of the present invention The nucleic acid construct of the present invention comprises a nucleic acid encoding a lentiviral vector, wherein the lentiviral vector comprises a 5'LTR, a packaging signal, and a 3'LTR in that order from its 5' end, and has a sequence of 10 to 65 bases derived from the nef gene adjacent to the 5' end of the 3'LTR. The nucleic acid construct of the present invention can be used to produce lentiviral vector particles. That is, by introducing the nucleic acid construct of the present invention into cells capable of producing lentiviral particles, lentiviral vector particles containing a viral genome transcribed from the nucleic acid construct can be produced. The nucleic acid construct of the present invention can produce lentiviral vector particles with a high viral titer. These lentiviral vector particles enable the introduction of a desired gene into cells with high efficiency and / or result in high expression of the introduced desired gene.

[0024] Lentiviruses are enveloped viruses with a positive-sense single-stranded RNA genome. The viral genome contains, from its 5' end, the 5' LTR, SD sequence, packaging signal, gag gene, poll gene, SA sequence, env gene, and 3' LTR. In the case of HIV-1, in addition to these elements, several accessory genes (vif, vpr, vpu, rev, tat, nef) are also included. Of these, the 5' LTR, packaging signal, and 3' LTR are essential to the viral genome in a lentiviral vector gene transfer system, and the nucleic acid construct of the present invention possesses all of these. Other elements can be deleted or rendered non-functional. Gene products such as gag, poll, and env, necessary for the production of lentiviral vector particles, can be supplied to cells by other means, such as plasmids containing these genes (called packaging plasmids).

[0025] Since the lentiviral packaging signal partially includes the gag gene sequence, it is not possible to completely remove the gag gene. Therefore, the nucleic acid construct of the present invention includes a partial sequence of the gag gene, preferably a sequence of 183 to 227 bp in length derived from the gag gene. Sequences of the aforementioned length are useful for efficient virus production and the expression of desired genes (Patent Document 1). Furthermore, since the risk of homologous recombination with the full-length gag gene used in virus production can be reduced, the present invention can provide a highly safe method for producing viral vectors. For example, the sequence derived from the nucleic acid encoding the gag protein is preferably 227 bp or 183 bp in length, and examples include the sequence described in Sequence ID No. 6 or 7, or a sequence in which one or several, for example, 1 to 9 bases are substituted, deleted, inserted, or added to this sequence.

[0026] In the lentiviral vector encoded by the nucleic acid construct of the present invention, the nef gene, which is unnecessary for the formation of lentiviral particles, has lost its function. The nucleic acid construct of the present invention has a deletion of a portion of the sequence derived from the nef gene adjacent to the upstream side of the 3' LTR, i.e., the 5' side, within the region encoding the lentiviral vector. Although not particularly limiting to the present invention, the sequence derived from the nef gene adjacent to the 5' side of the 3' LTR is 65 bases or less, preferably 30 bases or less, and more preferably 22 bases or less. Furthermore, the sequence derived from the nef gene is 10 bases or more, preferably 17 bases or more. In a further preferred embodiment of the present invention, the sequence includes the base sequence shown in Sequence ID No. 4.

[0027] The 5'LTR, 3'LTR, and packaging signal of the lentiviral vector contained in the nucleic acid construct of the present invention can be any of those capable of transcribing an RNA genome that can be packaged into lentiviral particles in cells into which the nucleic acid construct has been introduced.

[0028] LTRs and packaging signals usable in this invention include, for example, sequences derived from lentiviruses such as human immunodeficiency virus (HIV-1, HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), or canine arthritis encephalitis virus (CAEV). Lentiviral vectors can introduce genes into the nuclear genome regardless of mitosis in the cell into which the genes are introduced.

[0029] The LTR is functionally divided into three regions from the 5' end: U3, R, and U5. The U3 region has enhancer / promoter activity and directs the transcription of the viral genome (from the R region of the 5'LTR to the R region of the 3'LTR) by RNA polymerase II in the host cell. The 5'LTR used in the nucleic acid construct of the present invention may contain an exogenous enhancer / promoter for the virus from which the LTR originates. In one embodiment of the present invention, the U3 region or a portion of the 5'LTR is replaced with an exogenous enhancer / promoter. Similarly, the U3 region or a portion of the 3'LTR can be deleted, or the U3 region or a portion of the 3'LTR can be replaced with an exogenous enhancer / promoter.

[0030] Exogenous enhancers / promoters that substitute the U3 region of the LTR can be viral or mammalian, and constitutive, inducible, or tissue-specific enhancers / promoters can be used. For example, enhancers / promoters derived from viruses such as human cytomegalovirus (HCMV), Moloney's mouse sarcoma virus (MMSV), mouse stem cell virus (MSCV), Rous sarcoma virus (RSV), and spleen focus-forming virus (SFFV), as well as enhancers / promoters derived from mammalian genes such as β-actin, globin, elastase, albumin, α-fetoprotein, phosphoglycerate kinase (PGK), polypeptide chain elongation factor (EF1-α), and insulin, and even artificially constructed enhancers / promoters (e.g., MND promoter, CAG promoter) can be used. In a preferred embodiment of the present invention, a 5' LTR in which the U3 region is substituted with the HCMV immediate early enhancer / promoter is used. Here, the terms enhancer / promoter refer to sequences containing an enhancer region and / or a promoter region. Generally, the enhancer region and promoter region are collectively referred to as the promoter. Also, to distinguish them from the enhancer region, the promoter region alone is sometimes called the core promoter.

[0031] The 3' LTR used in this invention can be a sequence in which a mutation has been introduced into the U3 region to eliminate enhancer / promoter activity. As a result, when a viral vector particle containing a transcribed viral genome from this nucleic acid construct infects a cell, transcription from the R region is suppressed in the provirus formed when the double-stranded DNA generated from the viral genome is incorporated into the cell chromosome. A lentiviral vector with a mutated U3 region in the 3' LTR in this way is called a self-inactivating (SIN) vector. Mutations in the 3' LTR are introduced by base substitution or deletion. To express a desired gene in a provirus formed from a SIN vector, a promoter must be placed separately from the LTR. Furthermore, a separate promoter is also placed when expressing multiple desired genes. Such a promoter located between the 5' LTR and 3' LTR is sometimes called an internal promoter. The internal promoter can be derived from a virus, a mammalian gene, or an artificially constructed promoter. As internal promoters, enhancers / promoters or their promoter regions described herein, which can be used for substitution of the U3 region of the LTR, may be used. The internal promoter can be positioned between the packaging signal and the 3' LTR within the nucleic acid encoding the lentiviral vector.

[0032] The nucleic acid constructs of the present invention may include splicing donor (SD) sequences and / or splicing acceptor (SA) sequences. These sequences may be exogenous SD and / or SA sequences relative to the LTR, or exogenous SD and / or SA sequences relative to the internal promoter. Furthermore, the SD and SA sequences may be sequences of different origins. For example, 16S RNA from Simian virus (SV) 40, immediate early RNA from HCMV, and SD and SA sequences derived from the human hEF1α gene can be used [Proceedings of the National Academy of Sciences, Vol. 95, No. 1, pp. 219-223 (1998)]. In addition, SD or SA sequences in which mutations have been introduced into the consensus sequence to enhance or suppress splicing activity can also be used in the nucleic acid constructs of the present invention.

[0033] The foreign gene included in the nucleic acid construct of the present invention is a gene (also called a desired gene) that is to be expressed in cells infected with lentiviral vector particles produced using the nucleic acid construct of the present invention. This gene includes, for example, a protein-coding sequence, or a sequence that codes for intracellularly functional RNA such as tRNA or miRNA. Alternatively, a nucleic acid construct with multiple cloning sites may be produced, and then the desired gene may be inserted using these multiple cloning sites. Such nucleic acid constructs having multiple cloning sites instead of the desired gene are also included in the nucleic acid constructs of the present invention. These desired genes are usually positioned so that they can be transcribed by an internal promoter. That is, the desired gene is functionally linked to the internal promoter. Therefore, in the nucleic acid construct of the present invention, the desired gene or multiple cloning site functionally linked to the internal promoter is usually positioned between the internal promoter and the 3'-LTR.

[0034] The desired genes mentioned above may be intended for the prevention or treatment of diseases. Examples of genes include sequences useful for suppressing the transcription or expression of harmful gene products in the body (e.g., sequences encoding siRNA), sequences encoding proteins to replenish proteins that are deleted or have lost function in the body, and sequences that can modify or enhance the functions of cells. The present invention provides gene therapy in which cells into which foreign genes have been introduced using the nucleic acid construct of the present invention are introduced into the body. Examples of gene therapy include using the IL-2 receptor γ chain gene (X-linked severe combined immunodeficiency), the β-globin gene (β-thalassemia), the adenosine deaminase (ADA) gene (ADA deficiency), blood coagulation factor genes (hemophilia), and genes for antigen-recognizing receptors (cancer and viral infections).

[0035] In one aspect of the present invention, the desired gene included in the nucleic acid construct of the present invention includes a sequence encoding an oligomeric protein. Oligomer proteins include structural proteins, enzymes, transcription factors, receptors, and antibodies. In the present invention, the oligomeric protein may also be a cell surface protein (membrane protein), and the genes of antigen recognition receptors exemplified in the examples, such as T cell receptors (TCRs), are examples of desired genes.

[0036] In another aspect of the present invention, the desired gene included in the nucleic acid construct of the present invention is a chimeric antigen receptor (CAR) gene. The structure of a typical CAR consists of a single-chain variable fragment (scFv) that recognizes the surface antigen of target cells to be eliminated from the body, such as tumor cells, a transmembrane domain, and an intracellular domain that activates T cells. The intracellular domain of the TCR complex CD3ζ is preferably used as the intracellular domain. CARs with such a structure are called first-generation CARs. The gene for the single-chain antibody portion can be isolated, for example, from a hybridoma that produces a monoclonal antibody that recognizes the target antigen. T cells expressing a CAR can efficiently kill target cells by directly recognizing the surface antigen of target cells independently of the expression of major histocompatibility class I antigen on tumor cells and simultaneously activating T cells. Second-generation CARs, which ligate the intracellular domains of T cell costimulatory molecules such as CD28 or the tumor necrosis factor (TNF) receptor superfamily CD137 (4-1BB) or CD134 (OX40), as well as genes for third-generation CARs, which ligate these intracellular domains in tandem, can also be incorporated into the nucleic acid constructs of the present invention.

[0037] The nucleic acid constructs of the present invention may include post-transcriptional regulatory sequences (PREs), which are useful for enhancing the expression of desired genes. Examples of PREs used in the present invention include post-transcriptional regulatory sequences of herpes simplex virus, hepatitis B virus (HPRE), and woodchuck hepatitis virus (WPRE). The PREs include the coding region of the X protein, which has been linked to carcinogenicity. Mutant sequences in which the expression of the X protein from the PRE is suppressed can also be used in the nucleic acid constructs of the present invention. For example, a PRE in which a mutation causing a frameshift is introduced into the sequence coding for the X protein, or a PRE in which a stop codon that interrupts the translation of the X protein is inserted can be used. In one embodiment of the present invention, a PRE can be used in which the A of the start codon ATG of the X protein is taken as position 1, and a PRE containing one or two inserted bases that cause a frameshift between positions 6 and 7 can be used. Alternatively, a PRE in which positions 7 to 9 are replaced with a stop codon (e.g., TAA) can be used. WPREs are preferred for the nucleic acid constructs of the present invention. The WPRE may be one of those described in U.S. Patent No. 6,136,597, U.S. Patent No. 7,419,829, or Gene Therapy, Vol. 16, pp. 605–619 (2009), or a modification thereof. In one aspect of the present invention, a WPRE of the nucleotide sequence shown in SEQ ID NO: 8, a WPRE of the nucleotide sequence shown in SEQ ID NO: 9 or SEQ ID NO: 10 (WPRE2, WPRE3), or a WPRE of a nucleotide sequence in which one or more, for example, 1 to 9 nucleotides are substituted, deleted, inserted, or added to these sequences can be used. In one aspect of the nucleic acid construct of the present invention, the PR is located between the internal promoter (the gene if it has a desired gene, or the multicloning site if it has a multicloning site) and the 3' LTR sequence.

[0038] The nucleic acid construct of the present invention may include a Rev response element (RRE) and / or a central polyprint lact (cPPT). Examples of RREs include, but are not limited to, those located at positions 7622–8459 of the HIV NL4-3 genome (GenBank accession number: AF003887), or RREs derived from other strains of HIV or other retroviruses. The cPPT used in the present invention is not particularly limited, but examples include cPPTs derived from HIV-1, HIV-2, or SIV. For example, HIV-1 derived cPPTs are preferably used. In the nucleic acid construct of the present invention, the arrangement of both elements is not limited, but preferably between the packaging signal and the 3'LTR, or between the packaging signal and the internal promoter if the lentiviral vector has an internal promoter.

[0039] The nucleic acid construct of the present invention may include other elements outside the region encoding the lentiviral vector. For example, in the nucleic acid construct of the present invention in the form of a nucleic acid molecule such as a plasmid, other elements may be included on the 5' side of the 5' LTR and / or on the 3' side of the 3' LTR. In a preferred embodiment of the present invention, the nucleic acid construct of the present invention has the polyadenylation signal located on the 3' side of the region encoding the lentiviral vector, i.e., on the 3' side of the 3' LTR. Without particularly limiting the present invention, the distance between the 3' LTR and the polyadenylation signal is, for example, within 150 bases, preferably within 100 bases, and more preferably within 80 bases. The present invention can use polyadenylation signals that function in mammalian cells, such as polyadenylation signals derived from viruses such as SV40 virus and herpes simplex virus, or polyadenylation signals derived from growth hormone genes (bovine, human, etc.) or β-globin genes (human, rabbit, etc.), or artificially constructed polyadenylation signals.

[0040] The construct of the present invention, constructed as a plasmid, i.e., a lentiviral vector plasmid, has, in addition to the elements described above, a plasmid replication origin, a selection marker gene (drug resistance gene), and other elements. When the nucleic acid construct of the present invention is loaded onto a plasmid, two construction methods are possible: either aligning the transcription direction of the lentiviral genome with the direction of plasmid replication, or aligning it with the opposite direction. However, as far as the titer of the lentiviral vector is concerned, there is no significant difference between aligning and aligning.

[0041] (2) Method for producing lentiviral vector particles of the present invention The present invention includes a method for producing lentiviral vector particles, comprising the step of introducing the nucleic acid construct of the present invention into cells having the ability to produce lentiviral particles.

[0042] The nucleic acid constructs of the present invention can be introduced into cells using appropriate vectors, such as plasmid vectors, non-lentiviral viral vectors, or transposon vectors, so that they can stably exert their effects within cells. That is, the nucleic acid constructs of the present invention can be in the form of plasmids, viruses, or transposons. Furthermore, the nucleic acid constructs of the present invention can be incorporated into the chromosomal DNA of cells (for example, using genome editing technology). Alternatively, these nucleic acid constructs can be introduced directly into cells. Methods for introducing the nucleic acid constructs into cells directly or via plasmid vectors can be used, such as using carriers such as liposomes or ligand-polylysine, calcium phosphate methods, electroporation methods, or particle gun methods. The viral vectors are not particularly limited, and commonly known viral vectors used in gene transfer methods, such as adenovirus vectors, adeno-associated virus vectors, Simian virus vectors, vaccinia virus vectors, measles virus vectors, or Sendai virus vectors, can be used.

[0043] Cells capable of generating lentiviral particles are cells capable of producing proteins necessary for replication and packaging of the viral genome, in addition to the proteins that constitute the lentiviral particles. Examples of such proteins include the products of the gag gene, pol gene, env gene, and rev gene. For example, lentiviral vector particles can be produced by simultaneously or ad-hocly introducing the genes encoding the aforementioned proteins and the nucleic acid construct of the present invention into cells with high transfection efficiency, preferably human-derived cells (such as 293 cells or 293T cells), and culturing the resulting cells (also called lentiviral-producing cells) in an appropriate medium. The medium is not particularly limited, but known cell culture media such as DMEM, IMDM, Ham F12 medium, or modified versions thereof can be used. The medium may contain serum or be serum-free. Various commercially available media can also be used. The genes encoding the proteins necessary for the generation of lentiviral particles are introduced into cells, for example, by insertion onto one or more plasmids. Such plasmids for lentiviral vector particle production (called packaging plasmids) are widely available commercially and can be used in the method of the present invention. Lentiviral vector particles are typically released into the cell culture supernatant. The produced lentiviral vector particles contain transcripts from the nucleic acid constructs of the present invention. These lentiviral vector particles are included in the present invention as vectors for introducing desired genes into cells.

[0044] In a lentiviral vector, virus particles presenting a protein different from the Env protein of the base lentivirus can be produced on the surface. Such an operation is called pseudotyping. Pseudotyping is carried out, for example, by using lentivirus-producing cells into which a packaging plasmid containing an array encoding a protein desired to be presented on the virus particles is introduced. For pseudotyping, for example, packaging plasmids encoding Env proteins derived from Moloney murine leukemia virus (MMLV), gibbon ape leukemia virus (GaLV), vesicular stomatitis virus (VSV), feline endogenous virus, etc., or other proteins that can function as Env proteins can be used. Furthermore, by using packaging cells into which an enzyme gene involved in sugar chain synthesis or the like has been introduced, lentiviral vector particles having a protein that has undergone sugar chain modification on their surface can be produced.

[0045] After culturing the lentivirus-producing cells prepared by the above operation, the cell culture is centrifuged to collect the supernatant, and contaminants are removed by appropriate filtration operations to obtain lentiviral particles. Although the gene transfer can be carried out by contacting the cells directly with these crudely purified lentiviral particles, virus particles with higher purity may be prepared through known purification operations and used for the gene transfer operation.

[0046] The present invention provides a composition comprising the lentiviral vector particles of the present invention as an active ingredient together with pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are well known to those skilled in the art and include, for example, phosphate-buffered saline (e.g., 0.01 M phosphate, 0.138 M NaCl, 0.0027 M KCl, pH 7.4), aqueous solutions containing mineral salts such as hydrochloride, hydrobromide, phosphate, and sulfate, saline solution, glycol, or ethanol, and salts of organic acids such as acetate, propionate, malonate, and benzoate. Auxiliaries such as wetting agents or emulsifiers, and pH buffers may also be used. Examples of pharmaceutically acceptable excipients include those described in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). The composition may be in known forms suitable for parenteral administration, such as injection or infusion. Furthermore, formulation aids such as suspending agents, preservatives, stabilizers, and / or dispersants may be used, as well as preservatives to extend the shelf life during storage. The composition may also be in a dry form for reconstitution with a suitable sterile liquid before use.

[0047] (3) Gene transfer method of the present invention The gene transfer method of the present invention is characterized by comprising the step of infecting target cells with lentiviral vector particles obtained by the method for producing lentiviral vector particles of the present invention described in (2) above. The chromosomes of the target cells infected with the lentiviral vector particles have DNA incorporated that corresponds to the region sandwiched between the 5' LTR and 3' LTR on the nucleic acid construct of the present invention. In one embodiment of the present invention, this step is carried out in vitro.

[0048] In the method of the present invention, as target cells, cells derived from mammals, such as human-derived cells or non-human mammalian-derived cells such as monkeys, mice, rats, pigs, cows, dogs, etc. can be used. There is no particular limitation on the cells used in the method of the present invention, and any cells can be used. For example, cells collected, isolated, purified, or induced from body fluids, tissues, or organs such as blood (peripheral blood, cord blood, etc.), bone marrow, etc. can be used. Peripheral blood mononuclear cells (PBMCs), immune cells [T cells, dendritic cells, B cells, hematopoietic stem cells, macrophages, monocytes, NK cells, or blood cell lineage cells (neutrophils, basophils)], cord blood mononuclear cells, fibroblasts, preadipocytes, hepatocytes, skin keratinocytes, mesenchymal stem cells, adipose stem cells, various cancer cell lines, or neural stem cells can be used. In the present invention, the use of immune cells, precursor cells of immune cells (such as hematopoietic stem cells, lymphocyte precursor cells, etc.), or cell populations containing these is particularly preferred. T cells, which are representatives 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 precursor cells of T cells include PBMCs. Furthermore, NK cells, NKT cells, and their precursor cells can also be targeted by the method of the present invention. Examples of the above cells include those collected from a living body, those obtained by expanding and culturing them, those established as cell lines, those differentiated from pluripotent stem cells, etc., but are not limited thereto. When it is desired to transplant the produced gene-introduced cells or cells differentiated from the said cells into a living body, it is preferable to produce gene-introduced cells from cells collected from the living body itself or the same species of living body.

[0049] In the present invention, functional substances that improve the introduction efficiency can also be used in the step of infecting cells with lentiviral vector particles (for example, International Publication No. 95 / 26200, International Publication No. 00 / 01836). Examples of substances that improve introduction efficiency include substances that have activity to bind to lentiviral vector particles, such as fibronectin or fibronectin fragments. Preferably, fibronectin fragments having a heparin-binding site, such as the fragment commercially available as retronectin (Registered Trademark, CH-296, manufactured by Takara Bio Inc.), can be used. Commercially available lentiviral gene transfer aids may also be used.

[0050] In a preferred embodiment of the present invention, the functional substances may be used in a manner suitable for each substance. For example, retronectin can be used immobilized on a suitable solid phase, such as a container used for cell culture (plate, petri dish, flask, or bag, etc.) or a carrier (microbeads, etc.).

[0051] Gene-transformed cells obtained by the method of the present invention express the gene product encoded by the desired gene. Therefore, the gene-transformed cells of the present invention acquire new properties and / or functions resulting from the said gene product.

[0052] In one aspect of the present invention, gene-transformed cells obtained according to the present invention can be used as a therapeutic agent for diseases. The therapeutic agent contains the cells of the present invention, which can express gene products useful for treating diseases, as an active ingredient, and may further contain appropriate excipients. The excipients are not particularly limited as long as they are pharmaceutically acceptable, but examples include stabilizers, buffers, and isotonic agents. Diseases to which the cells of the present invention are administered are not particularly limited, as long as they are diseases that are sensitive to the cells, but examples include cancer [blood cancer (leukemia), solid tumors, etc.], inflammatory diseases / autoimmune diseases (asthma, eczema, etc.), hepatitis, and infectious diseases caused by viruses, bacteria, or fungi (influenza, AIDS, tuberculosis, coronavirus infection, VRE infection, deep-seated mycosis). Cells expressing TCRs or CARs that recognize antigens possessed by cells that are to be reduced or eliminated in the above diseases, i.e., tumor antigens, viral antigens, bacterial antigens, etc., are administered for the treatment of these diseases. Furthermore, the cells of the present invention can be used for purposes such as preventing infections after bone marrow transplantation or radiation therapy, and for donor lymphocyte transfusion aimed at achieving remission in relapsed leukemia. Therapeutic agents containing the cells of the present invention as an active ingredient can be administered parenterally, for example by injection or infusion, to the skin, muscle, subcutaneous, intraperitoneal, nasal, arterial, venous, tumor, or afferent lymphatic vessels.

[0053] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0054] Example 1: Preparation of a nef gene-derived sequence-reduced viral vector plasmid. First, the DNA of the MND promoter shown in Sequence ID No. 1 was synthesized and a recombinant plasmid was prepared by inserting it into the EcoRI-BamHI digest product of pLVpro-Promoterless-Km Vector (Takara Bio Inc., product code 6968). pLVpro-Promoterless-Km Vector is prepared by inserting the DNA of a lentiviral vector sequence, which has a 5' LTR in the 5' to 3' direction in which the U3 region is replaced with a CMV enhancer / promoter, a packaging signal, RRE, cPPT / CTS, a multi-cloning site, WPRE, and a 3' LTR (Δ3' LTR) with a portion of the U3 region deleted, into a plasmid containing the pUC plasmid replication origin and the kanamycin resistance gene. Next, the DNA of the sequence encoding ZsGreen1 from pLVpro-MSCV-ZsGreen1 Vector (manufactured by Takara Bio, product code 6965) was prepared, and this was inserted into the NotI-XhoI digest product of the recombinant plasmid to produce the pLVpro-MND-ZsGreen1 plasmid.

[0055] The base sequence of the 188 bp region of the HIV-1 nef gene-derived sequence contained in pLVpro-MND-ZsGreen1 that does not overlap with Δ3'LTR is described in Sequence ID No. 2. Next, as shown in Figure 1, plasmids A, B, C, and D were constructed by reducing the HIV-1 nef gene-derived sequence contained in pLVpro-MND-ZsGreen1 from 5' to 22 bp, 17 bp, 2 bp, and 0 bp, respectively. The HIV-1 nef gene-derived sequences contained in plasmids A and B are described in Sequence ID No. 3 and No. 4, respectively. The 16th base in Sequence ID No. 3 and the 11th base in Sequence ID No. 4 have A / G variations depending on the HIV-1 strain from which they are derived, but in the nef gene-derived sequences of plasmids A and B used in this example, the base in question is A. The nef gene-derived sequence of plasmid C is 5'-AC-3'.

[0056] Example 2 Preparation of Lentivirus Solutions Escherichia coli HST08 (Takara Bio Inc.) was transformed with pLVpro-MND-ZsGreen1 and plasmids A, B, C, and D prepared in Example 1. The plasmid DNA retained by these transformants was purified using NucleoSpin® Plasmid Midi (Mach Reiner Gel Inc.) and used as transfection DNA for the following procedure. Each of the prepared plasmids and a packaging plasmid (which confers transient expression of HIV-1-derived Gag, Pol, and Rev lentiviral proteins and VSV-G envelope protein to the host) were introduced into 293T cells (ATCC CRL-11268). Each of the resulting transduced cells was cultured, and the supernatant containing lentiviral particles with VSV-G envelopes was collected from each culture medium. The supernatant was filtered through a 0.45 μm filter (Milex HV, Millipore) and identified as virus solution LVpro-MND-ZsGreen1, solution A, solution B, solution C, and solution D.

[0057] Example 3: Infection of cells with a nef gene-derived sequence-reduced lentiviral vector The virus solution prepared in Example 2 was appropriately diluted and used to infect human T lymphocytic leukemia-derived cell line SupT1 cells (ATCC CRL-1942) once. Using a flow cytometer, the percentage of ZsGreen1-positive cells and the average fluorescence intensity in positive cells were measured 4 days after viral infection, and the viral titer of the supernatant prepared in Example 2 was calculated according to the following formula.

[0058] Virus titer (IFU / mL) = Number of infected cells × (Positivity rate % / 100) × Virus dilution ratio / Volume of virus at time of infection (mL)

[0059] Figure 2 shows a comparison of the average fluorescence intensity of each virus solution, with LVpro-MND-ZsGreen1 set as 100%, and Figure 3 shows a comparison of the virus titer. As shown in Figure 2, virus solutions A and B showed average fluorescence intensity equivalent to LVpro-MND-ZsGreen1, while virus solutions C and D showed lower average fluorescence intensity than LVpro-MND-ZsGreen1. Furthermore, as shown in Figure 3, an increase in virus titer was observed for virus solutions A and B, while a significant decrease was observed for virus solutions C and D.

[0060] Example 4 Optimization of the insertion site of the SV40 polyadenylation signal The pLVpro-MND-ZsGreen1 prepared in Example 1 was modified as follows. As shown in Figures 4 to 7, plasmid E was created by deleting the SV40 polyadenylation signal, plasmid F was created by moving the SV40 polyadenylation signal to a position 74 bases from the 3' side of the 3'LTR, and plasmid G was created by reversing the backbone (kanamycin resistance gene-origin region) of plasmid F.

[0061] Viral solutions were prepared for pLVpro-MND-ZsGreen1 and plasmids E, F, and G using the same method as in Example 2. The prepared viral solutions were designated as solution E, solution F, and solution G, respectively, and SupT1 cells were infected with these solutions at various dilution ratios. Cells were collected three days after viral infection, and the percentage of cells expressing the ZsGreen1 gene was measured using a flow cytometer. The viral titer was calculated using the values ​​where the ZsGreen1 positive rate was 1.0-20.0%, and the results are shown in Figure 8. As shown in Figure 8, the viral titer of viral solution E was equivalent to that of LVpro-MND-ZsGreen1, but an increase in viral titer was observed in viral solutions F and G.

[0062] The nef gene-derived sequence present in plasmid F was reduced to 22 bp, similar to plasmid A prepared in Example 1, to create plasmid pLVpro2-MND-ZsGreen1. The sequence of the prepared plasmid is shown in Sequence ID No. 5. Using this plasmid and pLVpro-MND-ZsGreen1, a virus solution was prepared in the same manner as in Example 2 (the virus solution derived from pLVpro2-MND-ZsGreen1 was designated as LVpro2-MND-ZsGreen1), and SupT1 cells were infected at various dilution ratios. Cells were collected four days after viral infection, and the percentage of ZsGreen1-expressing cells was measured using a flow cytometer. The viral titer was calculated using the measured values ​​where the ZsGreen1 positivity rate was 1.0-20.0%, and the results are shown in Figure 9. As shown in Figure 9, LVpro2-MND-ZsGreen1 showed an increase in viral titer compared to LVpro-MND-ZsGreen1.

[0063] Example 5 Modified pLVpro2 plasmids Plasmids were prepared by replacing the MND promoter of pLVpro-MND-ZsGreen1 prepared in Example 1 and pLVpro2-MND-ZsGreen1 prepared in Example 4 with the MSCV promoter (SEQ ID NO: 11), the EF1α promoter (SEQ ID NO: 12), and the EF1α-core promoter (SEQ ID NO: 13), respectively, which is the EF1α promoter with the exon intron sequence removed. The resulting plasmids were named pLVpro-MSCV-ZsGreen1, pLVpro-EF1α-ZsGreen1, pLVpro-EFS-ZsGreen1, pLVpro2-MSCV-ZsGreen1, pLVpro2-EF1α-ZsGreen1, and pLVpro2-EFS-ZsGreen1, respectively. Figure 10 shows the structure of the inserted DNA (lentiviral vector portion) contained in each plasmid.

[0064] Using the prepared plasmids, virus solutions were prepared in the same manner as in Example 2, and the prepared virus solutions were named LVpro-MNDp, LVpro-MSCVp, LVpro-EF1αp, LVpro-EF1α-corep, LVpro2-MNDp, LVpro2-MSCVp, LVpro2-EF1αp, and LVpro2-EF1α-corep, respectively. The viral titer of each virus solution was measured in the same manner as in Example 3. Figure 11 shows a comparison of viral titers when the viral titer of the LVpro-type virus solution is set to 100%. As shown in Figure 11, regardless of the type of promoter it carried, the LVpro2-type viruses (LVpro2-MNDp, LVpro2-MSCVp, LVpro2-EF1αp, LVpro2-EF1α-corep) showed a higher viral titer compared to the LVpro-type viruses.

[0065] Furthermore, for each of the eight types of virus solutions mentioned above, the viral titer was measured using the human fibrosarcoma-derived cell line HT1080 (ATCC CRL-121). The titer was measured in the same manner as described in Example 3, except that the cells were changed from SupT1 to HT1080 and the measurement of ZsGreen1-positive cells was performed 3 days after infection. Figure 12 shows a comparison of viral titers when the LVpro type viral titer is set to 100%. As shown in Figure 12, even when using HT1080 cells, the LVpro2 type viruses equipped with each promoter showed a higher viral titer compared to the LVpro type viruses.

[0066] Furthermore, the viral solutions LVpro-MNDp, LVpro-MSCVp, LVpro2-MNDp, and LVpro2-MSCVp were diluted to various concentrations and used to infect peripheral blood mononuclear cells (PBMCs) isolated from human peripheral blood using RetroNectin (Takara Bio). Six days after viral infection, the cells were collected and stained with APCcy7-labeled anti-Human CD8 antibody (Becton Dickinson) and Pecy7-labeled anti-Human CD4 antibody (BioLegend). Subsequently, CD8-positive cells, CD4-positive cells, and ZsGreen1-positive cells were measured using a flow cytometer. The percentage of ZsGreen1-positive cells in CD8-positive cells and CD4-positive cells, respectively, is shown in Figure 13. LVpro2-type viruses showed higher infection efficiency compared to LVpro-type viruses in both CD8-positive and CD4-positive cells.

[0067] Example 6 Plasmid DNA was prepared by substituting the ZsGreen1 encoding sequence of pLVpro-MND-ZsGreen1, prepared in Example 1, and pLVpro2-MND-ZsGreen1, prepared in Example 4, with the encoding sequence of a chimeric antigen receptor (CD19 CAR) that specifically recognizes CD19 [J. Immunol., Vol. 32, No. 7, pp. 689-702 (2009)]. These were named pLVpro-MND-CD19 CAR and pLVpro2-MND-CD19 CAR, respectively. The structure of the inserted DNA held by each plasmid is shown in Figure 14.

[0068] Using the prepared plasmids, viral solutions were prepared in the same manner as in Example 2, and the resulting viral solutions were named LVpro-MND-CD19-28Z and LVpro2-MND-CD19-28Z. The prepared viral solutions were diluted as appropriate and used to infect SupT1 cells once. Cells were collected 4 days after viral infection and stained with PE-labeled Whitlow / 218 Linker (E3U7Q) antibody (Cell Signaling). Next, the percentage of cells expressing anti-CD19-CAR was measured using a flow cytometer, and the viral titer of the supernatant prepared according to the following formula was calculated.

[0069] Virus titer (IFU / mL) = Number of infected cells × (Positivity rate % / 100) × Virus dilution ratio / Volume of virus at time of infection (mL)

[0070] Figure 15 shows the results of calculating the viral titer using measurements where the percentage of anti-CD19-CAR expressing cells was 1.0–20.0%. As shown in Figure 15, the viral titer was elevated for the vector LVpro2-MND-CD19-28Z compared to LVpro-MND-CD19-28Z.

[0071] Furthermore, PBMCs were infected with LVpro-MND-CD19-28Z and LVpro2-MND-CD19-28Z diluted at various ratios using RetroNectin. Cells were collected 6 days after viral infection and stained with PE-labeled Whitlow / 218 Linker (E3U7Q) antibody, APCcy7-labeled anti-Human CD8 antibody, and Pecy7-labeled anti-Human CD4 antibody. Anti-CD19-CAR expressing cells, CD8-positive cells, and CD4-positive cells were measured using a flow cytometer, and the percentage of cells expressing anti-CD19-CAR calculated from these results is shown in Figure 16. It was revealed that the LVpro2 type virus had a higher infection efficiency in CD8-positive and CD4-positive cells compared to the LVpro type virus.

[0072] The present invention provides a nucleic acid construct useful for producing high-titer lentiviral vector particles, lentiviral vector particles obtained using the nucleic acid construct, a method for producing genetically modified cells using the vector particles, and the genetically modified cells. These nucleic acid constructs, lentiviral vector particles, the method for producing genetically modified cells, and the genetically modified cells are extremely useful for protein production, disease treatment by gene therapy, and research and testing for these purposes.

[0073] SEQ ID NO:1: MND promoter SEQ ID NO:2: pLVpro-MND-ZsGreen1 nef sequence SEQ ID NO:3: plasmid A nef sequence SEQ ID NO:4: plasmid B nef sequence SEQ ID NO:5: pLVpro2-MND-ZsGreen1 SEQ ID NO:6: truncated gag sequence SEQ ID NO:7: truncated gag sequence SEQ ID NO:8: mutated WPRE sequence SEQ ID NO:9: WPRE2 sequence SEQ ID NO:10: WPRE3 sequence SEQ ID NO:11: MSCV promoter SEQ ID NO:12: EF1α promoter SEQ ID NO:13: EF1α-core promoter

Claims

1. A nucleic acid construct comprising nucleic acid encoding a lentiviral vector, wherein the lentiviral vector comprises, in order from its 5' end, a 5' LTR, a packaging signal, and a 3' LTR, and adjacent to the 5' end of the 3' LTR is a sequence of 10 to 65 bases derived from the nef gene.

2. The nucleic acid construct according to claim 1, wherein the sequence derived from the nef gene includes the sequence shown in Sequence ID No.

4.

3. The nucleic acid construct according to claim 1 or 2, wherein the U3 region or a portion thereof of the 3'LTR is deleted.

4. A nucleic acid construct according to any one of claims 1 to 3, wherein a polyadenylation signal is present on the 3' side of the 3'-LTR.

5. A nucleic acid construct according to any one of claims 1 to 4, wherein the U3 region or a portion thereof of the 5'LTR is replaced with an exogenous promoter.

6. A nucleic acid construct according to any one of claims 1 to 5, comprising an internal promoter between the packaging signal and the 3'LTR.

7. The nucleic acid construct according to claim 6, comprising a multi-cloning site or an exogenous gene functionally linked to the internal promoter between the internal promoter and the 3'LTR.

8. The nucleic acid construct according to claim 6, comprising a PRE between the internal promoter and the 3'LTR.

9. The nucleic acid construct according to claim 7, comprising a PRE between an exogenous gene functionally linked to a multicloning site or internal promoter and the 3' LTR.

10. Lentivirus-producing cells into which the nucleic acid construct according to any one of claims 1 to 9 has been introduced.

11. A method for producing lentiviral vector particles, comprising the steps of culturing lentiviral-producing cells according to claim 10, and collecting lentiviral vector particles from the cell culture obtained in the first step.

12. A method for introducing a gene into a target cell, comprising the steps of: producing lentiviral vector particles by the method described in claim 11; and infecting target cells with the lentiviral vector particles obtained in the above step.

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