WO2023038055A1 - Acide nucléique pour la production de vecteurs rétroviraux - Google Patents

Acide nucléique pour la production de vecteurs rétroviraux Download PDF

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

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

Abstract

La présente invention concerne une construction d'acide nucléique destinée à produire un vecteur rétroviral et comprenant chacune des séquences suivantes dans cet ordre à partir de l'extrémité 5' : (a) une séquence 5'LTR (longue répétition terminale) dérivée d'un rétrovirus comprenant une séquence promoteur exogène ; (b) une séquence signal d'encapsidation (ψ) dérivée d'un rétrovirus ; (c) une séquence dérivée d'un acide nucléique codant pour une protéine gag d'une longueur de 183 à 227 pb ; (d) une séquence désirée ou un site de multiclonage ; et (e) une séquence 3'LTR dérivée d'un rétrovirus.
PCT/JP2022/033543 2021-09-08 2022-09-07 Acide nucléique pour la production de vecteurs rétroviraux WO2023038055A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002516065A (ja) * 1997-12-22 2002-06-04 オックスフォード バイオメディカ（ユーケイ）リミテッド ウマ伝染性貧血ウイルス（ｅｉａｖ）系
WO2021070956A1 (fr) * 2019-10-11 2021-04-15 タカラバイオ株式会社 VECTEUR D'EXPRESSION D'ARNsi

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002516065A (ja) * 1997-12-22 2002-06-04 オックスフォード バイオメディカ（ユーケイ）リミテッド ウマ伝染性貧血ウイルス（ｅｉａｖ）系
WO2021070956A1 (fr) * 2019-10-11 2021-04-15 タカラバイオ株式会社 VECTEUR D'EXPRESSION D'ARNsi

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Gene transfer using lentiviral vector. Product guide.", TAKARA-BIO CO., 1 September 2020 (2020-09-01), XP093044068, Retrieved from the Internet <URL:https://catalog.takara-bio.co.jp/CONTENTS/catalog_request/pdf/lenti.pdf> [retrieved on 20230503] *
CUI Y., IWAKUMA T., CHANG L.-J.: "CONTRIBUTIONS OF VIRAL SPLICE SITES AND CIS-REGULATORY ELEMENTS TO LENTIVIRUS VECTOR FUNCTION.", JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 73., no. 07., 1 July 1999 (1999-07-01), US , pages 6171 - 6176., XP000882588, ISSN: 0022-538X *
MAKI, IZUMI ET AL.: "2P-0376 Development of lentiviral vector for highly effective and safe gene therapies", PROCEEDINGS OF 43RD ANNUAL MEETING OF THE MOLECULAR BIOLOGY SOCIETY OF JAPAN MBSJ 2020; ONLINE; DECEMBER 2-4, 2020, vol. 43, 19 November 2020 (2020-11-19) - 4 December 2020 (2020-12-04), pages 1331, XP009544296 *
POESCHLA E ET AL.: "Identification of a human immunodeficiency virus type 2 (HIV-2) encapsidation determinant and transduction of nondividing human cells by HIV-2-based lentivirus vectors", JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 72, no. 8, 1 August 1998 (1998-08-01), US , pages 6527 - 6536, XP002141655, ISSN: 0022-538X *

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