WO2011062155A1 - ACIDE NUCLÉIQUE CODANT POUR UNE PROTÉINE Gag MUTANTE - Google Patents

ACIDE NUCLÉIQUE CODANT POUR UNE PROTÉINE Gag MUTANTE Download PDF

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WO2011062155A1
WO2011062155A1 PCT/JP2010/070359 JP2010070359W WO2011062155A1 WO 2011062155 A1 WO2011062155 A1 WO 2011062155A1 JP 2010070359 W JP2010070359 W JP 2010070359W WO 2011062155 A1 WO2011062155 A1 WO 2011062155A1
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nucleic acid
amino acid
retrovirus
protein
derived
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淳 駒野
英人 蝶野
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • the present invention relates to a nucleic acid useful for preparing a retroviral vector encoding a mutant Gag protein, a retroviral vector-producing cell having the nucleic acid, a packaging plasmid having the nucleic acid, a packaging cell having the nucleic acid, and the The present invention relates to a packaging plasmid or a retroviral vector production system containing the packaging cell, and a retroviral vector produced by the retroviral vector-producing cell.
  • Viral vectors are a technology widely used from basic to clinical in the field of gene therapy. Gene transfer methods using retroviral vectors are currently the most widely used for gene therapy because they can integrate the gene of interest into the host chromosome and expect permanent expression of the gene of interest in the host cell. It is a gene transfer method.
  • Retrovirus vectors are obtained as recombinant virus particles in the culture supernatant by culturing retrovirus vector-producing cells.
  • retrovirus particles tend to be deactivated when suspended in a medium, and are also easily deactivated by a concentration operation. Therefore, in order to efficiently introduce a gene using a retroviral vector, it is necessary to improve the titer (number of virus particles per unit volume) and infectivity of the retroviral vector produced by the retroviral vector-producing cells. It is said that.
  • a human cytomegalovirus-derived promoter having a promoter activity stronger than that of a terminal repeat sequence (LTR) is used as an attempt to improve the titer of a retroviral vector produced by a retroviral vector-producing cell.
  • LTR terminal repeat sequence
  • Non-patent Document 1 a high-titer retrovirus has been developed by using a Gag protein having a sequence encoding a pleckstrin homology domain (PH domain) of phospholipase C- ⁇ 1 added to the N-terminus in a retroviral vector production system.
  • PH domain pleckstrin homology domain
  • retroviral vectors obtained using this method have been found to have improved infectivity compared to retroviral vectors obtained with a retroviral vector production system in which no mutation has been introduced into the Gag protein. Absent.
  • the problem to be solved by the present invention is to provide a retroviral vector production system capable of performing gene transfer more efficiently and a retroviral vector produced by the production system.
  • the present inventors retrovirally replace a nucleic acid containing a nucleic acid sequence encoding a mutant Gag protein in which an amino acid residue is added and / or inserted into the N-terminal and / or MA domain of the amino acid sequence of a retrovirus-derived Gag protein.
  • retroviral vector retroviral particles
  • the present inventors have found that the infectivity of the thus obtained retrovirus to cells is higher than that of the retrovirus obtained by the conventional method, thereby completing the present invention.
  • a first aspect of the present invention is a nucleic acid encoding a mutant Gag protein obtained by adding and / or inserting one or more amino acid residues to the amino acid sequence of a retrovirus-derived Gag protein, (A) a nucleic acid sequence encoding a mutant Gag protein obtained by adding an amino acid sequence containing a lipid modification signal to the N-terminus of a retrovirus-derived Gag protein; and (b) an amino acid sequence of a matrix domain of the retrovirus-derived Gag protein.
  • the present invention relates to a nucleic acid comprising a nucleic acid sequence selected from the group consisting of nucleic acid sequences encoding a mutant Gag protein into which one or more amino acid residues are inserted.
  • Examples of the lipid modification signal in the first aspect of the present invention include a myristoylation signal and a double lipid modification signal of myristoylation and palmitoylation, and the retrovirus-derived Gag protein is derived from a lentivirus or an oncoretrovirus The Gag protein is exemplified.
  • the second aspect of the present invention relates to a retrovirus vector-producing cell having the nucleic acid of the first aspect of the present invention.
  • the third aspect of the present invention relates to a packaging plasmid having the nucleic acid of the first aspect of the present invention.
  • the fourth aspect of the present invention relates to a packaging cell having the nucleic acid of the first aspect of the present invention.
  • the fifth aspect of the present invention comprises (A) the packaging plasmid of the third aspect of the present invention or the packaging cell of the fourth aspect of the present invention; and (B) a terminal repeat sequence derived from a retrovirus, packaging.
  • the present invention relates to a retrovirus vector production system comprising a transfer vector containing a signal and a cloning site into which a foreign gene can be inserted.
  • the sixth aspect of the present invention relates to a retroviral vector produced by the retroviral vector-producing cell of the second aspect of the present invention.
  • the retroviral vector of the sixth aspect of the present invention is exemplified by a lentiviral vector or a retroviral vector based on Moloney murine leukemia virus (MMLV).
  • MMLV Moloney murine leukemia virus
  • a nucleic acid useful for the production of a retroviral vector a packaging cell containing the nucleic acid, a packaging plasmid, and a retroviral vector producing cell, a retroviral vector production system comprising the packaging cell or the packaging plasmid, and A retroviral vector produced by the retroviral vector production system is provided. Since the retroviral vector produced by the retroviral vector production system using the nucleic acid of the present invention can efficiently perform gene introduction, it is useful for production of pharmaceuticals and gene introduction into target cells for gene therapy. It is.
  • retrovirus refers to a generic name of RNA viruses belonging to the Retroviridae family whose genome is composed of RNA and has a life cycle for converting the RNA into DNA, and includes oncoretrovirus and lentivirus.
  • oncoretrovirus is Moloney murine leukemia virus (MMLV).
  • MMLV Moloney murine leukemia virus
  • lentivirus include human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV).
  • Gag protein refers to a protein encoded by a retrovirus gag gene.
  • the Gag protein is expressed as a single precursor protein from a gene on proviral DNA inserted into the host chromosome by the retrovirus.
  • a long precursor in which three types of viral enzymes (protease, reverse transcriptase, and integrase) are fused to the C-terminus of the Gag protein is also expressed. These precursors localize to the cell membrane when their N-terminus is myristoylated, and budding as viral particles including the RNA genome. Thereafter, it is processed by a protease derived from a retrovirus to become mature virus particles.
  • Gag protein is composed of three types of proteins, matrix (MA), capsid (CA), and nucleocapsid (NC) in this order from the N-terminal side of the precursor protein. Among these, there is a myristoylation signal in the M domain of MA.
  • a lipid modification signal refers to an amino acid sequence that commands modification of a protein with lipids such as fatty acids and isoprenoids.
  • lipid modification signals for example, myristoylation signals, palmitoylation signals, myristoylation and palmitoylation double lipid modification signals, O-acylation signals, and isoprenylation signals are known.
  • the myristoylation signal is an amino acid sequence that directs myristoylation of a protein and usually consists of 6 to 9 amino acids.
  • Protein myristoylation is a lipid modification of a protein in which a myristoyl group derived from myristic acid, a saturated fatty acid having 14 carbon atoms, is covalently bonded to the N-terminal glycine residue of the protein. It is estimated that this modification occurs in .5%.
  • Virus-derived proteins having a myristoylation signal include Gag proteins of various retroviruses such as human immunodeficiency virus (HIV) and Moloney murine leukemia virus (MMLV), HIV Nef protein, MMLV tyrosine protein kinase, Rous sarcoma virus (RSV) pp60v-src, human hepatitis B virus (HBV) pre-S1 core protein, poliovirus core protein VP4 are known.
  • HSV human immunodeficiency virus
  • MMLV Moloney murine leukemia virus
  • RSV Rous sarcoma virus
  • HBV Rous sarcoma virus
  • HBV Rous sarcoma virus
  • HBV Rous sarcoma virus
  • HBV hepatitis B virus
  • poliovirus core protein VP4 poliovirus core protein VP4
  • Non-virus-derived proteins having a myristoylation signal include protein kinases such as Src family tyrosine kinases, phosphatases such as calcineurin B, guanine nucleotide binding proteins such as G ⁇ proteins, and EF-handed Ca 2+ binding proteins such as recoverins.
  • Structural proteins such as myristoylated alanine rich C kinase substrate (MARKKS) are known.
  • N-terminal start methionine followed by glycine is essential.
  • serine or threonine is present at the 6th amino acid from the N-terminus
  • lysine or arginine is present at the 7th and / or 8th amino acid.
  • Whether a myristoylation signal is present in the amino acid sequence of a protein can be predicted using, for example, “The MYR Predictor” (http://mendel.imp.ac.at/myristate/SUPLpredictor.htm). is there.
  • the palmitoylation signal is an amino acid sequence that directs palmitoylation of a protein, and it is known that a cysteine residue is essential.
  • the consensus sequence of palmitoylation signals other than cysteine residues is not known, but for example, the algorithm “CSS-Palm version 2.0” [Protein Engineering Design and Selection (Protein Eng. Des. Sel.), Vol. 21, No. 11, pp. 639-644 (2008)], it is possible to predict whether a palmitoylation signal is present in the amino acid sequence of a protein.
  • Proteins having a palmitoylation signal include G ⁇ s subunit, ⁇ subunit of guanine nucleotide-binding protein such as G ⁇ q subunit (G ⁇ subunit), growth-related protein-43 (GAP-43), calcium channel ⁇ 2a subunit, synapse Post-thickening protein-95 (PSD-95), G protein signaling regulator-4 (RGS4), and the like are known.
  • the double lipid modification signal of myristoylation and palmitoylation is an amino acid sequence that commands the double lipid modification of protein myristoylation and palmitoylation.
  • Proteins having a dual lipid modification signal of myristoylation and palmitoylation include Src family tyrosine kinases such as Yes, Fyn, Lyn, Lck, Hck, Fgr and Yrk, G ⁇ i1 subunit, G ⁇ o subunit, and G ⁇ z. G ⁇ subunit, A kinase anchor protein (AKAP-18), endothelial nitric oxide synthase (eNOS) and the like are known.
  • the Src family tyrosine kinases and G ⁇ subunits having the double lipid modification signal of myristoylation and palmitoylation have the sequence of Met-Gly-Cys at the N-terminus, and this sequence is myristoylation of these proteins. It is said that the sequence is necessary and sufficient for palmitoylation. Whether or not the myristoylation and palmitoylation double lipid modification signals are present in the amino acid sequence of the protein can be predicted by, for example, the above-mentioned “The MYR Predictor” and “CSS-Palm version 2.0”.
  • nucleic acid of the present invention is a nucleic acid comprising a nucleic acid sequence encoding a mutant Gag protein obtained by adding and / or inserting one or more amino acid residues to the amino acid sequence of a retrovirus-derived Gag protein.
  • a nucleic acid consisting of a nucleic acid sequence and / or a nucleic acid consisting of a nucleic acid sequence encoding a mutant Gag protein in which one or more amino acid residues are inserted into the amino acid sequence of the MA domain of a retrovirus-derived Gag protein are preferred.
  • a mutant Gag protein obtained by adding an amino acid residue to the amino acid sequence of a retrovirus-derived Gag protein means an N-terminal methionine residue originally possessed by the Gag protein or 1 to 5 residues from the N-terminus.
  • insertion of an amino acid residue means a case where a new amino acid residue is added between amino acid residues at a specific position.
  • nucleic acid sequence encoding a mutant Gag protein may be a part of a nucleic acid sequence encoding a fusion protein of a mutant Gag protein and a Pol protein. preferable. That is, a nucleic acid containing a nucleic acid sequence encoding a mutant Gag-Pol fusion protein is one of the preferred embodiments of the nucleic acid of the present invention.
  • the retrovirus-derived Gag protein is not particularly limited as long as it is derived from a retrovirus.
  • a protein is preferably exemplified.
  • the mutant Gag protein in the present invention has a lipid modification signal in its N-terminal region.
  • the lipid modification signal present in the N-terminal region of the added amino acid sequence and the myristoylation signal inherent in the Gag protein loses its function as a result of the addition of the amino acid sequence to the N-terminus.
  • the myristoylation originally possessed by the retrovirus-derived Gag protein is used.
  • An amino acid residue may be inserted into the MA domain so as to retain the signal, or an amino acid residue may be inserted so that a lipid modification signal that the retrovirus originally does not have exists in the N-terminal region.
  • the mutant Gag protein in the present invention preferably has a region (basic region) rich in basic amino acid residues in the vicinity of the N-terminus thereof.
  • the basic region is not particularly limited, but a basic region near the N-terminus of the retrovirus-derived Gag protein can be used.
  • the “mutant Gag protein in which an amino acid sequence containing a lipid modification signal is added to the N-terminus of a retrovirus-derived Gag protein” in the present invention is preferably the N-terminus of the amino acid sequence of the retrovirus-derived Gag protein. 1 or more residues, preferably 6 to 100 residues, more preferably 7 to 50 residues, still more preferably 7 to 30 Examples thereof include mutant Gag proteins to which residues, more preferably 8 to 25 amino acid residues, are added.
  • the “mutant Gag protein in which one or more amino acid residues are inserted into the amino acid sequence of the matrix domain of the retrovirus-derived Gag protein” includes the amino acid sequence of the MA domain of the retrovirus-derived Gag protein, One or more residues, preferably 1 to 100 residues, more preferably 6 to 100 residues, still more preferably 7 to 50 residues, still more preferably 7 to 30 residues, still more preferably 8 to 25 residues And a mutant Gag protein having a lipid modification signal in the N-terminal region thereof.
  • the mutant Gag protein in the present invention is not particularly limited as long as it has addition and / or insertion of an amino acid residue at the above position.
  • the mutant Gag protein is not limited to the above position in the amino acid sequence of the retrovirus-derived Gag protein.
  • those having an amino acid sequence in which amino acid deletion, substitution, insertion, addition or the like has occurred in the range of several amino acid residues are also included. Therefore, the homology between the amino acid sequence of the mutant Gag protein and the amino acid sequence of the retrovirus-derived wild-type Gag protein is preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, and even more preferably. 95% or more.
  • homology is defined as NCBI-BLAST (Basic Local Alignment Search Tool) (http://blast.ncbi.nlm.nih.gov/Blast.cgi), FSA-BLAST (http: //www.fsa -blast.org/), WU-BLAST (http://blast.wustl.edu/), FASTA, Clustal W, Clustal X, etc.
  • NCBI-BLAST Basic Local Alignment Search Tool
  • FSA-BLAST http: //www.fsa -blast.org/
  • WU-BLAST http://blast.wustl.edu/
  • FASTA Clustal W
  • Clustal X etc.
  • the one or more amino acid residues to be added and / or inserted are not particularly limited as long as they constitute an amino acid sequence including a lipid modification signal when added to the N-terminus of a Gag protein. And an amino acid sequence including a linker sequence.
  • an amino acid sequence including a linker sequence When inserted into the matrix domain of a Gag protein, it is inserted so that the myristoylation signal originally possessed by the retrovirus-derived Gag protein is retained as it is, or a lipid modification signal that the retrovirus does not originally have is N-terminal region
  • the lipid modification signal used in the present invention may be virus-derived or non-virus-derived.
  • the lipid-modified signal derived from a virus used in the present invention is not particularly limited to the present invention, but is derived from a myristoylation signal derived from an oncoretrovirus such as MMLV, and a lentivirus derived from HIV-1 or SIV.
  • a myristoylation signal is preferably exemplified.
  • the non-virus-derived lipid modification signal used in the present invention is not particularly limited, and examples thereof include lipid modification signals possessed by eukaryotic proteins.
  • the non-virus-derived lipid modification signal used in the present invention is not particularly limited, but a non-virus-derived myristoyl signal and a double lipid modification signal of myristoylation and palmitoylation are preferred. Illustrated.
  • the present invention is not particularly limited, but myristoylation derived from Src family tyrosine kinase, G ⁇ protein, or nitric oxide synthase And the dual lipid modification signal of palmitoylation is preferred.
  • the amino acid sequences of these lipid modification signals can be appropriately modified and used in the present invention.
  • the myristoylation and palmitoylation double lipid modification signal sequences are modified so that the palmitoylation signal is deleted, It can also be used as an activation signal.
  • a novel lipid modification signal may be designed based on the sequence information of a known lipid modification signal.
  • a high titer virus vector can be obtained by using the nucleic acid of the present invention for the production of a retrovirus vector. Furthermore, the retroviral vector produced using the nucleic acid of the present invention exhibits higher infectivity (infection efficiency) than that obtained by the conventional method.
  • the titer of the retroviral vector produced using the nucleic acid of the present invention can be calculated from the quantification result of the structural protein of the viral particle by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • a commercially available p24 ELISA kit ZeptoMetrix
  • it can be measured by quantifying the produced viral RNA molecules by real-time RT-PCR.
  • the infectivity of a retroviral vector is determined by, for example, preparing a retroviral vector holding a marker gene such as a luciferase gene or a fluorescent protein gene and introducing it into a target cell, and the resulting expression of the marker gene is This can be confirmed by examining the appearance rate of the observed cells.
  • a marker gene such as a luciferase gene or a fluorescent protein gene
  • the nucleic acid of the present invention includes genes encoding other retroviral structural proteins such as the pol gene and env gene in addition to the above-described gene encoding the mutant Gag protein (mutant gag gene). Also good.
  • the mutant gag gene and the pol gene may be contained in the nucleic acid of the present invention as a mutant gag-pol gene encoding a mutant Gag-Pol fusion protein.
  • the env gene is a gene encoding an envelope (Env protein). Since the Env protein specifically recognizes a receptor present on the target cell membrane, it is one of the important factors that determine the cells that can be infected by the virus. It is.
  • the env gene used in the present invention one derived from the same virus as the gag gene can be used, but it is used for the production of a pseudo-type vector having a host range different from the retrovirus from which the gag gene is derived.
  • an env gene derived from a virus different from the gag gene can also be used.
  • the env gene derived from a virus different from the gag gene include the env gene derived from gibbon leukemia virus (GaLV) and the G glycoprotein (VSV-G) gene derived from vesicular stomatitis virus.
  • Retroviral vector-producing cell of the present invention is a recombinant virus produced by gene recombination technology based on an oncoretrovirus, a lentivirus or the like belonging to a retrovirus.
  • Particles retroviral vector particles
  • oncoretrovirus vector include a vector based on Moloney leukemia virus (MMLV).
  • MMLV Moloney leukemia virus
  • lentiviral vectors include vectors based on human immunodeficiency virus type 1 (HIV-1), vectors based on simian immunodeficiency virus (SIV), and the like.
  • oncoretrovirus vectors and lentivirus vectors pseudotyped using Env proteins derived from viruses different from Gag proteins and Pol proteins are also included in the “retrovirus vectors” described herein.
  • the present invention is used to create a recombinant retroviral vector that is deficient in replication.
  • a retrovirus packaging cell in which a gene encoding a retrovirus structural protein such as a gag-pol gene or an env gene has been incorporated into a chromosome in advance is packaged with a foreign gene.
  • a method of producing by introducing a transfer vector carrying a signal, and a packaging plasmid having a gene encoding a retrovirus structural protein such as a gag-pol gene or an env gene in a cell having no retroviral structural protein At the same time, there can be mentioned a method of producing the aforementioned transfer vector by transfection.
  • a method for producing the retroviral vector of the present invention a method comprising the step of preparing the retroviral producer cell of the present invention using the packaging cell of the present invention described later, and the packaging plasmid of the present invention described later are used. And a method including the step of preparing the retrovirus-producing cell of the present invention using the method.
  • retrovirus vector-producing cells are often prepared using 293 cell lines, 293T cell lines, and 293FT cell lines with high transfection efficiency as hosts.
  • a retroviral vector-producing cell refers to a cell having all the elements necessary for production of a recombinant retroviral vector.
  • the elements necessary for the production of the recombinant retroviral vector are not particularly limited as long as the mutant gag gene is included, but in addition, pol gene, env gene, LTR, packaging signal, Examples include foreign genes that are desired to be retained in a recombinant retroviral vector, and examples include accessory genes such as the rev gene.
  • the retroviral vector-producing cell of the present invention has the nucleic acid of the present invention and can produce a retroviral vector.
  • the retroviral vector-producing cell of the present invention contains a mutant Gag protein, a mutant Gag-Pol protein, an Env protein encoded by the nucleic acid of the present invention, and an LTR, a packaging signal, and a foreign gene.
  • a transcription product or the like is expressed, and a retrovirus vector in which the transcription product is packaged can be budding.
  • retroviral vector-producing cells of the present invention have the nucleic acid of the present invention, a large amount of retroviral vector particles can be produced.
  • the production amount of virus particles such as retrovirus vector particles can be confirmed by, for example, quantifying the structural protein (eg, p24) of virus particles by enzyme-linked immunosorbent assay (ELISA) or quantifying the amount of genomic RNA by RT-PCR. I can do it.
  • ELISA enzyme-linked immunosorbent assay
  • I can do it.
  • an HIV-1 p24 antigen ELISA kit manufactured by ZeptoMetrix
  • ZeptoMetrix can be used.
  • the nucleic acid sequence encoding the mutant Gag protein (mutant gag gene) and the env gene are not adjacent to each other. It is preferable.
  • a retroviral vector-producing cell in which a sequence encoding a mutant Gag-Pol protein and an env gene are integrated at different positions on a chromosome, a plasmid having a sequence encoding a mutant Gag-Pol protein, and env Retroviral vector-producing cells into which both different plasmids containing genes have been introduced are suitable as the retroviral vector-producing cells of the present invention.
  • the transfer vector used for the preparation of the retrovirus vector-producing cells of the present invention is not particularly limited, but for example, retroviruses such as pBabe, pLXIN (Clontech), and pDON-AI-2 (Takara Bio).
  • retroviruses such as pBabe, pLXIN (Clontech), and pDON-AI-2 (Takara Bio).
  • a transfer vector in which a foreign gene is inserted into a plasmid vector for vector expression is exemplified.
  • lentivirus-derived transfer vectors include pLVX series (Clontech) and pLenti6 / V5 (Invitrogen).
  • any gene desired to be introduced into the cell can be selected.
  • an enzyme related to a disease to be treated a protein (for example, an enzyme, an antibody, a receptor, a structural protein, a growth factor, etc.), an antisense nucleic acid, a ribozyme, RNA for inducing RNA interference
  • a false primer, a T-cell receptor gene that specifically recognizes a tumor antigen, or the like can be used.
  • the transfer vector may have an appropriate marker gene that enables selection of the transfected cell.
  • the marker gene for example, a drug resistance gene that confers resistance to antibiotics on a cell, a gene that encodes a fluorescent protein, or a reporter gene that can distinguish a cell into which a gene has been introduced by detecting enzyme activity can be used.
  • a foreign gene to be introduced into a cell by a retroviral vector can be used by inserting it into a transfer vector for expression of a retroviral vector under the control of an appropriate promoter, for example, an LTR promoter or a foreign promoter. .
  • an appropriate promoter for example, an LTR promoter or a foreign promoter.
  • other regulatory elements that cooperate with the promoter and transcription initiation site to achieve transcription of the foreign gene such as an enhancer sequence, may be present in the transfer vector.
  • the introduced foreign gene can contain a terminator sequence downstream thereof.
  • the introduced foreign gene may be natural or artificially produced, or may be one in which DNA molecules of different origins are linked by ligation or other means known in the art. .
  • Packaging plasmid of the present invention has the nucleic acid of the present invention, and is introduced into a cell together with the transfer vector by a method such as transfection to produce the retroviral vector of the present invention. Can be used for cell preparation.
  • a typical packaging plasmid has a nucleic acid sequence encoding a Gag-Pol protein placed under the control of a promoter. Some have genes of env genes and other virus-derived accessory proteins. Preferred examples of the packaging plasmid of the present invention include those containing the nucleic acid sequence of the present invention encoding the mutant Gag-Pol protein and not containing the env gene from the viewpoint of preventing the appearance of self-replicating retroviral particles. Is done.
  • a cell producing the retroviral vector of the present invention can be prepared by transfecting a host cell with an env vector containing a transfer vector and an env gene.
  • the rev gene is further mounted on any of the above vectors, or a rev vector containing the rev gene is separately transfected into the host cell together with the above vectors. Also good.
  • Packaging cell of the present invention has the nucleic acid of the present invention, and the transfer vector is introduced into the cell by a method such as transfection, whereby the retroviral vector of the present invention. Production cells are prepared.
  • the packaging cell refers to a cell that lacks a nucleic acid having a packaging signal ( ⁇ sequence) necessary for packaging of virus particles and has a gene encoding a retrovirus structural protein.
  • a packaging cell has a gag gene, a pol gene, and an env gene, and one or more expression cassettes that can express Gag, Pol, and Env proteins.
  • a packaging cell containing a gag gene and a pol gene and not containing an env gene, such as a GP2-293 packaging cell is known. When such a cell is used, for example, pVSV-G is used.
  • Retroviral vectors can be produced by co-transfecting a plasmid that expresses the Env protein with a transfer vector.
  • a cell host for producing a packaging cell a cell having high gene transfer efficiency by transfection or a cell having high expression of a structural gene after transfer is preferably used.
  • mouse fibroblast NIH3T3 strain or human fetus Renal cell line 293 can be used.
  • a general gene introduction method such as a calcium phosphate method, a transfection method using a liposome, or an electroporation method can be used.
  • the resulting packaging cells are subjected to limiting dilution, and then a transfer vector is introduced into each clone, the titer of the virus produced is measured, and the cell producing the highest titer virus is selected and cloned. It is preferable to do.
  • Retroviral vector production system of the present invention includes the packaging plasmid of the present invention or the packaging cell of the present invention, and a transfer vector comprising a cloning site into which a foreign gene can be inserted. .
  • the transfer vector into which the foreign gene can be inserted which is included in the retroviral vector production system of the present invention, is not particularly limited, but general transfer such as pLXRN, pLNHX, and pDON-AI-2. Vectors can be used.
  • a typical transfer vector has a foreign gene, a retrovirally derived terminal repeat (LTR), a packaging signal, a primer binding site essential for reverse transcription, and no other retrovirus-derived gene.
  • the U3 region of the 5 ′ LTR of the transfer vector introduced into the packaging cell of the present invention may be replaced with a strong promoter such as a cytomegalovirus-derived promoter in order to increase the transcription efficiency of the target gene. Good.
  • the transfer vector may have an appropriate marker gene that allows selection of the transfected cell.
  • the packaging cell expresses SV40 large T antigen
  • the use of a transfer vector combined with the SV40 replication origin can increase the copy number in the packaging cell, and the produced retrovirus.
  • the titer of the vector can be improved.
  • a desired foreign gene is inserted into a transfer vector capable of inserting a foreign gene contained in the retroviral vector production system of the present invention, and the resulting transfer vector is represented by the present invention.
  • the retrovirus vector-producing cells of the present invention can be prepared by transfecting the above packaging cells.
  • the packaging cell of the present invention does not contain an env gene
  • the retroviral vector-producing cell of the present invention is transformed by transfecting the packaging cell with an envelope plasmid capable of expressing the Env protein together with the transfer vector.
  • a REV plasmid containing the rev gene may be transfected into a packaging cell together with each of the above vectors.
  • a desired foreign gene is inserted into a transfer vector into which a foreign gene can be inserted, which is included in the retroviral vector production system of the present invention, and the obtained transfer vector and the packaging plasmid of the present invention are appropriately used.
  • the retroviral vector-producing cells of the present invention can be prepared by co-transfection into a suitable host cell.
  • the packaging plasmid of the present invention does not contain the env gene
  • the retrovirus vector-producing cell of the present invention is prepared by transfecting a host cell with an envelope plasmid capable of expressing Env protein together with the above vector. I can do things.
  • a REV plasmid containing the rev gene may be transfected into a host cell together with each of the above vectors.
  • Retroviral vector of the present invention is produced by the retroviral vector-producing cell of the present invention. Since the retroviral vector of the present invention exhibits high infectivity, it is useful, for example, for gene transfer into target cells for gene therapy, production of pharmaceuticals and the like.
  • the retrovirus vector of the present invention is not particularly limited to the present invention, but a lentivirus vector or an oncoretrovirus vector based on MMLV is preferred.
  • retroviral vectors for example, a retroviral vector carrying a marker gene such as a luciferase gene is introduced into a target cell, and the resulting rate of appearance of cells in which the marker gene is observed is examined. Can be confirmed.
  • a marker gene such as a luciferase gene
  • the retroviral vector of the present invention comprises a matrix (MA), a capsid (CA), and a nucleocapsid (NC) that are produced by processing the mutant Gag protein encoded by the nucleic acid of the present invention during the maturation process of the virus particle. It has RNAs containing LTR, packaging signal, and gene of interest derived from transfer vectors.
  • MA matrix
  • CA capsid
  • NC nucleocapsid
  • Example 1 Construction of a packaging plasmid (gag-pol expression vector) for producing a lentiviral vector that expresses a N-terminal-modified Gag protein (and Gag-Pol protein) was performed as follows.
  • a plasmid pgag- constructed by codon-optimizing the gag-pol gene of the natural gag-pol expression vector of HIV-1 without changing the amino acid sequence encoding so that expression in human cells is optimized.
  • pol [[Journal of General Virology, Vol. 89, pp. 1949-1955 (2008)], and the sequence of the pgag-pol gene contained in pgag-pol is shown in SEQ ID NO: 1 in the Sequence Listing. ] was designated as wild type gag-pol expression vector plasmid WT LV_Gag.
  • myristoylation palmitoylation signal (myristoylation and palmitoylation double lipid modification signal) consisting of 11 amino acid residues derived from Lyn and a linker sequence for a total of 24 amino acid residues (SEQ ID NO: 2) of the wild type Gag protein
  • SEQ ID NO: 2 For the vector plasmid Lyn MyPa LV_Gag that expresses a mutant Gag protein (mutant Gag-Pol protein) added in place of two residues from the N-terminal side, the nucleic acid sequence of the gag-pol gene in the plasmid WT LV_Gag It was constructed by modification.
  • the nucleic acid sequence of the gag-pol gene in the plasmid Lyn MyPa LV_Gag is shown in SEQ ID NO: 3.
  • substitution of the third cysteine from the N-terminus to the alanine of the mutant Gag protein expressed by the plasmid Lyn MyPa LV_Gag results in two types of myristoylation and palmitoylation of Lyn.
  • a vector plasmid Lyn My LV_Gag expressing a mutant Gag protein (mutant Gag-Pol protein) in which a heavy lipid modification signal was converted to a myristoylation signal was constructed.
  • the amino acid sequence of the N-terminal 24 residues of the mutant Gag protein expressed by Lyn My LV_Gag is shown in SEQ ID NO: 4, and the nucleic acid sequence of the gag-pol gene in Lyn My LV_Gag is shown in SEQ ID NO: 5.
  • a mutant Gag protein obtained by adding a total of 24 amino acid residues (SEQ ID NO: 6) consisting of an HIV myristoylation signal and a linker sequence in place of amino acids corresponding to 2 residues from the N-terminal side of the wild-type Gag protein was added.
  • the total of 24 amino acid residues (SEQ ID NO: 8) consisting of the vector plasmid HIV-1 My LV_Gag to be expressed and the myristoylation signal of MMLV and the linker sequence (SEQ ID NO: 8) are replaced with 2 amino acids from the N-terminal side of the wild-type Gag protein.
  • the vector plasmid MLV My LV_Gag expressing the added mutant Gag protein was constructed by modifying the nucleic acid sequence of the gag-pol gene in the plasmid WT LV_Gag.
  • the nucleic acid sequence of the gag-pol gene in the plasmid HIV-1 My LV_Gag and the nucleic acid sequence of the gag-pol gene in the MLV My LV_Gag are shown in SEQ ID NO: 7 and SEQ ID NO: 9, respectively.
  • Table 1 shows the N-terminal amino acid sequence of the Gag protein expressed by each of the above gag-pol expression vector plasmids.
  • the second glycine from the N-terminus is myristoylated by post-translational modification.
  • the third cysteine from the N-terminus is palmitoylated by post-translational modification.
  • Example 2 The virus solution was prepared according to the following procedure. 5 ⁇ 10 5 293FT (Invitrogen) was added to each well of a 6-well culture plate (NUNK) and cultured for 24 hours. As the medium, RPMI 1640 medium (Sigma) containing 10% fetal bovine serum (Japan Visylum) and 10 mM HEPES was used.
  • each gag-pol expression vector plasmid prepared in Example 1 0.65 ⁇ g of lentiviral vector plasmid plenti-luciferase (Invitrogen, transfer vector having a luciferase gene), VSVG envelope expression vector plasmid pVSV-G (Clontech) 0.4-0.8 ⁇ g and Rev expression vector plasmid pRevpac [Molecular Cell Biology, Vol. 15, No. 12, pp. 5197-5207 (2004)] Lipofectamine 2000 (Invitrogen) ), And 4-6 hours later, the cells were subcultured to a new 6 cm petri dish (Nunk Co.) and further cultured. 48 hours after transfection, the cell culture supernatant was collected and filtered with a 0.45 ⁇ m filter, and this filtrate was used as a virus supernatant in the following experiments.
  • lentiviral vector plasmid plenti-luciferase Invitrogen, transfer vector having a luciferase gene
  • virus production was confirmed by quantifying the retrovirus structural protein p24 in the cell culture supernatant using the p24 ELISA kit (ZeptoMetrix). The infection efficiency was confirmed by adding 800 ⁇ L of virus supernatant to 293FT cells seeded on a 24-well plate, and measuring luciferase activity 48 hours later using Steady-Glo Luciferase assay system (Promega). The results are shown in FIG. In FIG. 1, “virus production amount” is expressed as a multiple of the concentration of p24 in the cell culture supernatant obtained using plasmid WT LV_Gag, and “gene transfer efficiency” is a virus vector prepared using plasmid WT LV_Gag. Was expressed as a multiple of the luciferase activity when the cells were infected.
  • the use of a gag-pol expression vector plasmid that expresses a mutant Gag protein in the preparation of a virus is compared to the case where a gag-pol expression vector plasmid that expresses a wild-type Gag protein is used.
  • the virus production increased by 4 times or more and the infection efficiency improved by 11 times or more.
  • the present invention is useful for gene introduction into target cells for the production of pharmaceuticals and gene therapy.
  • SEQ ID NO: 1 A gag-pol gene encoding a wild type Gag protein derived from HIV. SEQ ID NO: 3; A mutant gag-pol gene in plasmid Lyn MyPa LV Gag. SEQ ID NO: 4; An amino acid sequence of N-terminal 24 residues of mutant Gag protein expressed by plasmid Lyn My LV_gag. SEQ ID NO: 5; A mutant gag-pol gene in plasmid Lyn My LV_Gag. SEQ ID NO: 7; A mutant gag-pol gene in plasmid HIV-1 My LV_Gag. SEQ ID NO: 9; A mutant gag-pol gene in plasmid MLV My LV_Gag.
  • SEQ ID NO: 11 An amino acid sequence of N-terminal 35 residues of mutant Gag protein expressed by plasmid Lyn MyPa LV_Gag. SEQ ID NO: 12; An amino acid sequence of N-terminal 35 residues of mutant Gag protein expressed by plasmid Lyn My LV_Gag. SEQ ID NO: 13; An amino acid sequence of N-terminal 35 residues of mutant Gag protein expressed by plasmid HIV-1 My LV_Gag. SEQ ID NO: 14; An amino acid sequence of N-terminal 35 residues of mutant Gag protein expressed by plasmid MLV My LV_Gag.

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Abstract

L'invention concerne un acide nucléique codant pour une protéine Gag mutante qui est obtenu par ajout et/ou insertion d'un ou de plusieurs résidus d'acides aminés dans la séquence d'acides aminés d'une protéine Gag dérivée d'un rétrovirus. L'acide nucléique est caractérisé en ce qu'il contient une séquence d'acides nucléiques qui est choisie dans le groupe constitué par (a) une séquence d'acides nucléiques codant pour une protéine Gag mutante qui est obtenue après ajout d'une séquence d'acides aminés qui contient un signal de lipidation à la terminaison N d'une protéine Gag dérivée d'un rétrovirus et (b) une séquence d'acides nucléiques codant pour une protéine Gag mutante qui est obtenue par insertion d'un ou de plusieurs résidus d'acides aminés dans la séquence d'acides aminés du domaine de matrice d'une protéine Gag dérivée d'un rétrovirus. L'acide nucléique codant pour une protéine Gag mutante est utile pour le transfert de gènes dans une cellule cible pour la fabrication de produits pharmaceutiques ou pour une thérapie génique.
PCT/JP2010/070359 2009-11-19 2010-11-16 ACIDE NUCLÉIQUE CODANT POUR UNE PROTÉINE Gag MUTANTE WO2011062155A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04500308A (ja) * 1989-05-17 1992-01-23 リサーチ・コーポレーション・テクノロジーズ・インコーポレーテッド 組換え型生成物のレトロウィルス媒介分泌
EP2080768A1 (fr) * 2008-01-21 2009-07-22 Freie Universität Berlin Procédé d'étude de la libération de particule de virus

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04500308A (ja) * 1989-05-17 1992-01-23 リサーチ・コーポレーション・テクノロジーズ・インコーポレーテッド 組換え型生成物のレトロウィルス媒介分泌
EP2080768A1 (fr) * 2008-01-21 2009-07-22 Freie Universität Berlin Procédé d'étude de la libération de particule de virus

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MERVIS RJ ET AL.: "The gag gene products of human immunodeficiency virus type 1: alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors", J VIROL, vol. 62, no. 11, 1988, pages 3993 - 4002 *
SATO I ET AL.: "Differential trafficking of Src, Lyn, Yes and Fyn is specified by the state of palmitoylation in the SH4 domain", J CELL SCI, vol. 122, 1 April 2009 (2009-04-01), pages 965 - 975 *

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