WO2012121071A1 - Vecteur adénoviral dans lequel les va-arn ne sont pas exprimés - Google Patents

Vecteur adénoviral dans lequel les va-arn ne sont pas exprimés Download PDF

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WO2012121071A1
WO2012121071A1 PCT/JP2012/055027 JP2012055027W WO2012121071A1 WO 2012121071 A1 WO2012121071 A1 WO 2012121071A1 JP 2012055027 W JP2012055027 W JP 2012055027W WO 2012121071 A1 WO2012121071 A1 WO 2012121071A1
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vector
gene
rna
rnas
cells
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水口　裕之
和史 形山
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国立大学法人大阪大学
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    • 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
    • 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
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10321—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to a highly safe gene therapy vector and an adenovirus vector that can be effectively used in life science research.
  • the present invention also relates to an adenovirus vector production / amplification cell capable of producing and / or amplifying the adenovirus vector.
  • Ad vectors Adenoviral vectors
  • Ad vectors have the highest gene transfer efficiency among existing gene therapy vectors and can be transferred to a wide range of cell types with a wide range of infections, so they are used in approximately 25% of gene therapy clinical research worldwide.
  • Ad vectors When an Ad vector is administered to a living body, (1) an innate immune reaction elicited by a vector particle constituent protein or vector genomic DNA occurs within several hours after the administration of the vector, and (2) derived from a foreign gene product and virus introduced by the vector. An acquired immune response to the protein is induced within a few days. Therefore, the development of Ad vectors that can avoid these immune reactions is urgently needed to develop highly safe gene therapy methods.
  • Ad vector development aimed at avoiding innate immune reactions:
  • various inflammatory mediators such as IL-6, IL-12, TNF, RANTES, MIP-2, and IFN- ⁇ / ⁇ / ⁇ are produced within 3 hours of administration.
  • Over 90% of systemically administered Ad vectors migrate to the liver (parenchymal cells and non-parenchymal cells including Kupffer cells), but many inflammatory cytokines are secreted primarily in the spleen, not in the liver. Therefore, an Ad vector that suppresses migration to the spleen has low production of inflammatory cytokines.
  • Non-patent Document 1 Koizumi et al. , 2007
  • IL-6 is hardly produced in the triple-modified Ad vector in which the binding properties to CAR, integrin, and heparan sulfate are deleted (Non-patent Document 2: Koizumi et al., 2006).
  • Non-Patent Document 1 Non-Patent Document 2
  • Ad vector development aimed at avoiding acquired immune reactions: When an Ad vector is administered to a living body, it usually shows transient gene expression for several weeks to several months. However, immunodeficient mice show gene expression over a long period of time from several months to more than one year (in some cases, a lifetime), so that transgenic cells are eliminated by the action of acquired immune responses centered on cellular immunity. This is considered to be a cause of transient expression of the Ad vector.
  • Conventional Ad vectors are designed to eliminate viral protein production by removing the E1 gene region essential for virus growth and viral protein production. There is a slight production of protein, which can be the target of the acquired immune system. The immunological properties of the foreign gene product introduced by the vector also affect the activation of the acquired immune system.
  • a gutted Ad vector has been developed in which all viral genomes other than the viral origin of replication and the region necessary for packaging have been removed.
  • this vector When this vector is used, long-term gene expression is observed even in normal mice (Non-patent Document 3: Dudley et al., 2004; Non-patent Document 4: Palmer et al., 2003).
  • the gutted Ad vector is a big problem that mass production is difficult and that a helper virus necessary for vector production is mixed, which is a big obstacle in drug development that should ensure uniformity.
  • an anti-Ad antibody is produced in an individual administered with an Ad vector, the effect is attenuated by systemic administration of the vector after the second time.
  • Non-patent literature 5 Roberts et al., 2006; Non-Patent Document 6: Sakurai et al., 2003).
  • VA-RNAs Virus-associated RNAs
  • VA-RNA I and / or VA-RNA II Virus-associated RNAs encoded by the Ad vector are recognized by innate immune receptors in the cytoplasm, and then induce interferon production, intracellular signals
  • IPS-1 interferon-beta promoter stimulator ⁇ 1
  • VA-RNAs It has been reported that it plays an important role in the immune reaction caused by the Ad vector (Non-patent Document 7: Yamaguchi et al., 2010).
  • VA-RNAs are untranslated RNA polymerase III transcripts having a length of about 160 bases.
  • VA-RNA I having a large transcription amount reaches ⁇ 10 8 molecules per cell during Ad replication.
  • VA-RNA I plays a central role in inhibiting activation of protein kinase R (protein kinase R: PKR) as an antiviral response of host cells during Ad replication, and in the absence of VA-RNA I It is known that PKR is activated and phosphorylation of the translation initiation factor eIF-2 ⁇ is induced, resulting in inhibition of viral mRNA translation.
  • protein kinase R protein kinase R
  • Ad in which the VA-RNAs gene has been mutated in viral therapy using restricted growth type Ad
  • Patent Documents 1 and 2 a virus for achieving selective replication in tumor cells is shown for the use of Ad to exert a cytocidal effect.
  • the virus lacks the VA-RNAs gene and has a genetic mutation that selectively expresses one or more genes in the E1a group, E1b group, and E4 group in tumor cells.
  • the promoter that controls one or more genes of the E1a group, the E1b group, and the E4 group has a mutation.
  • Ad from which VA-RNAs and the E1 gene region have been removed is not disclosed.
  • there is no report that the VA-RNAs of the Ad vector have been modified for the purpose of avoiding the side effects of the Ad vector.
  • RNA interference RNA interferance: RNAi
  • target therapeutic
  • siRNA double-stranded RNA
  • RNAi small interfering RNA
  • VA-RNAs remarkably suppress the RNAi effect by shRNA
  • VA-RNA I inhibits RNAi effect by competing with exportin5 for nuclear export of pre-miRNA and shRNA precursor, and binds to Dicer It has been reported that the function is inhibited, and as a result, the production of siRNA and mature miRNA is inhibited (Non-patent document 8: Andersson, MG et al., 2005, Non-patent document 9: Lu, S). . Et al., 2004).
  • the specific suppression of target gene expression by RNAi is one of the major issues in conducting research using RNAi, such as how to introduce a siRNA-expressing vector into a target cell.
  • MicroRNA plays an important role in the control of gene expression.
  • miRNA is transcribed from DNA as Pri-miRNA in the nucleus and becomes a hairpin double-stranded RNA (dsRNA) precursor. dsRNA moves into the cytoplasm, and mature miRNA is produced by the action of Dicer. The generated miRNA is incorporated into a RISC (RNA-induced silencing complex) complex and involved in gene function control.
  • RISC RNA-induced silencing complex
  • An object of the present invention is to provide an Ad vector that is highly safe and exhibits a gene transfer efficiency equal to or higher than that of a conventional Ad vector, that is, an Ad vector that can be effectively used in gene therapy and life science research. . Furthermore, it is an object to provide an Ad vector production / amplification cell capable of producing and / or amplifying the Ad vector.
  • the present inventors have solved the above problems by using an Ad vector in which the VA-RNAs gene is modified so that VA-RNAs are not expressed in the Ad vector genome.
  • the present invention was completed.
  • the above-mentioned problem is caused by a cell in which the E1 gene product used for the production and / or amplification of the Ad vector is constitutively expressed and the gene is introduced so that the VA-RNAs of the Ad genome are constitutively expressed.
  • the present invention was completed.
  • this invention consists of the following. 1. An Ad vector, wherein the E1 gene region is deleted from the Ad genome, and the VA-RNAs gene is modified so that VA-RNAs are not expressed. 2. 2. The Ad vector according to item 1, wherein the VA-RNAs gene is a VA-RNA I gene and / or a VA-RNA II gene. 3. 3.
  • the shRNA expression or miRNA expression vector contains a sequence capable of generating a desired siRNA or mature miRNA.
  • Ad vector production capable of amplifying the Ad vector according to any one of 1 to 8 above, wherein a part or all of the nucleotide sequence constituting the VA-RNAs gene of the Ad genome is introduced. -Cells for amplification. 10.
  • a method for producing an Ad vector comprising introducing the Ad vector genomic DNA sequence for producing the Ad vector according to any one of 1 to 8 above into the cell according to 9 or 10 above. 12 9. An RNAi induction method using the Ad vector according to item 7 or 8. 13. A kit comprising an Ad vector plasmid for producing the Ad vector according to any one of 1 to 8 above, and the Ad vector production / amplification cell according to 9 or 10 above. 14 A kit comprising the Ad vector according to any one of 1 to 8 above and the Ad vector production / amplification cell according to 9 or 10 above.
  • the Ad vector in which the VA-RNAs gene of the present invention is modified can suppress the Ad vector genomic DNA amplification in cells except for special cells, and can effectively introduce a desired gene into the cells.
  • conventional Ad vectors lack the E1 gene region, they are theoretically produced and / or amplified only in special cells that express the E1 gene product.
  • vector genomic DNA is amplified independently of the E1 gene in certain cells.
  • transcription of the viral protein coding gene remaining in the vector genome occurs independently of the E1 gene.
  • Amplification of the Ad vector genome independent of the E1 gene also increases the amount of viral protein expression that adversely affects cells.
  • the Ad vector with the modified VA-RNAs gene of the present invention has markedly suppressed replication of the Ad vector genome, the generation of CPE and the presentation of viral antigens are suppressed as a result, and inflammation and unnecessary immune reactions are suppressed. Is done. Therefore, the Ad vector in which the VA-RNAs gene of the present invention is modified can be said to be a vector whose safety has been drastically increased while maintaining the characteristics of an Ad vector with good gene transfer efficiency.
  • the gene expression period can be expected to be extended.
  • the RNAi effect induced by the shRNA expression Ad vector was not as high as expected.
  • the Ad vector in which the VA-RNAs gene of the invention is modified RNAi is effectively induced without suppressing the generation of mature miRNA or siRNA.
  • the Ad vector modified with the VA-RNAs gene of the present invention is useful as a gene transfer tool for improving the safety of gene therapy and life science research, and developing an shRNA-expressing Ad vector with high knockdown effect of the target gene can do.
  • an Ad vector in which the VA-RNAs gene of the present invention is modified can be prepared from an Ad vector genomic DNA sequence in which the VA-RNAs gene is modified.
  • Ad vectors can be amplified.
  • FIG. 4 is a photograph showing production and amplification of a conventional FG-Ad vector and an Ad ⁇ VR vector of the present invention in HEK293 cells. Amplification of the FG-Ad vector is observed, but amplification of the Ad ⁇ VR vector is not observed.
  • Example 1 It is the figure which confirmed the expression induction of VA-RNAVAI in VR293 cells.
  • Example 2 It is a photograph figure which shows production and amplification of Ad ⁇ VR vector in VR293 cells.
  • Example 2 It is the photograph which confirmed amplification of Ad ⁇ VR vector by PCR method.
  • Example 1 It is the photograph which confirmed the expression of VA-RNA II and VA-RNA II for the FG-Ad vector and the Ad ⁇ VR vector of the present invention. In Ad ⁇ VR vector, expression of VA-RNA I and VA-RNA II was not observed.
  • Example 2 It is the figure which confirmed the gene transfer efficiency in A549 cell and SK * HEP-1 cell about Ad (DELTA) VR vector.
  • Example 4 It is the figure which confirmed the expression of Ad-derived mRNA by the FG-Ad vector and the Ad ⁇ VR vector of the present invention in HUVEC cells. In the figure, simply showing Ad means FG-Ad. (Experimental example 4) It is a figure which shows the process which acts on the gene expression suppression of shRNA and VA-RNAs. Example 3 It is a figure which shows the plasmid for constructing shRNA expression Ad vector.
  • A) is pAdHM4-U6-shLuc-CG (FG-Ad-shLuc) containing VA-RNAs
  • B) is pAd ⁇ VR-U6-shLuc-CG (Ad ⁇ ) in which the expression of VA-RNAs is suppressed.
  • Example 3 It is a figure which shows the structure of VR-shLuc).
  • Example 3 It is the figure which confirmed the relative luciferase activity when FG-Ad-shLuc or Ad (DELTA) VR-shLuc was transfected into the SK * HEP1-Luc cell.
  • Example 3 It is the figure which confirmed the relative luciferase activity when FG-Ad-shLuc or Ad (DELTA) VR-shLuc was transfected into the SK * HEP1-Luc cell.
  • Ad means an adenovirus
  • Ad vector means a vector containing characteristic gene information of Ad.
  • Ad vector plasmid refers to a plasmid encoding an Ad vector genome.
  • Ad genome refers to the entire genetic information of Ad, that is, all of the DNA sequence included in Ad
  • Ad vector genome refers to the entire genetic information of Ad vector, that is, the DNA sequence included in Ad vector. Say everything.
  • the Ad vector of the present invention is characterized in that the E1 gene region of the Ad genome is deleted and the VA-RNAs gene is modified so that VA-RNAs are not expressed.
  • the Ad that can be used in the Ad vector of the present invention is not particularly limited as long as it can achieve the function as a vehicle for inserting a target DNA sequence into various types of cells in vivo or in vitro.
  • each of Ad of type 2, type 5, type 11 and type 35 using human as a host, monkey Ad, mouse Ad, dog Ad, sheep Ad and avian Ad other than human as host.
  • the deletion of the E1 gene region means that the DNA sequence encoding the E1 gene in the Ad genome is completely deleted.
  • the E1 gene region specifically corresponds to positions 342 to 3523 in the human type 5 Ad genome (GenBank Accession Number: M73260, M29978), for example.
  • the Ad vector of the present invention requires that the VA-RNAs gene is modified so that VA-RNAs are not expressed in addition to the deletion of the E1 gene region.
  • the modification of the VA-RNAs gene may be any modification as long as VA-RNAs are not expressed, and is not particularly limited. If VA-RNAs are not expressed, for example, a part or all of the DNA sequence encoding VA-RNAs may be deleted, or any of the DNA sequences encoding VA-RNAs is substituted. Or may be added.
  • VA-RNAs gene means VA-RNA I gene and / or VA-RNAVAII gene. “VA-RNAs do not express” may mean that only one of VA-RNA-I and VA-RNA II does not express.
  • the modification of the VA-RNAs gene may be at least one modification selected from the following 1) to 4), for example.
  • it may be a deletion of part or all of the consensus sequence of the internal promoter region of the VA-RNA II gene, deletion of part or all of the consensus sequence of the internal promoter region of the VA-RNA II gene, etc. .
  • Ad is human type 5 Ad
  • the region of the DNA sequence encoding VA-RNAs is 10620 to 11038 (SEQ ID NO: 1) in the human type 5 Ad genome (GenBank Accession No: M73260, M29978)
  • the region of the DNA sequence encoding VA-RNA I is 10620-1079 (SEQ ID NO: 2)
  • the region of the DNA sequence encoding VA-RNA II is 10876-11038 of the human type 5 Ad genome ( SEQ ID NO: 3).
  • the region that may be deficient is, for example, the following (a), (b), (c), (d), (e), (b) and (c), ( Combinations of b) and (e), (c) and (d), and (d) and (e) can be mentioned.
  • the region where the gene may be deficient is not limited to the above combination, but is mutated so that VA-RNAs are not transcribed to RNA polymerase III by deleting the sequence containing the internal promoter of VA-RNAs. May be added.
  • 10620-1079 deletion of all genes of VA-RNA I.
  • the Ad vector of the present invention may lack, for example, the E3 gene region in addition to the above-mentioned E1 region gene.
  • the E3 gene region refers to a region corresponding to 28133-30808 or 27865-30995 in the human type 5 Ad genome (GenBank Accession No .: M73260, M29978).
  • the deletion of the E3 gene region may be a partial or complete deletion of the DNA sequence encoding the E3 gene.
  • a foreign gene expression cassette can be inserted into a region lacking the E1 gene or a region lacking the E3 gene. Any gene that can be expressed as a foreign gene can be inserted.
  • the Ad vector in which the VA-RNAs gene of the present invention is modified can be used, for example, as a gene therapy vector or a vector as a gene introduction tool in life science research.
  • the present invention also extends to vectors for gene therapy and vectors for gene transfer tools.
  • a vector for a gene therapy vector or a gene introduction tool for example, it can be used as an expression vector for shRNA or miRNA.
  • RNAi effect induced by the shRNA expression Ad vector is not as high as expected, considering the high gene expression efficiency of the Ad vector. It is done.
  • VA-RNAs remarkably suppress the RNAi effect by shRNA (Non-Patent Documents 8 and 9).
  • VA-RNA I also inhibits the export of pre-miRNA and shRNA precursors by competing with exportin5 and inhibits its function by binding to Dicer, resulting in the generation of mature miRNA and siRNA. Has been reported to inhibit.
  • Ad vector in which the VA-RNAs gene of the present invention is modified does not compete with exportin5 for nuclear export of shRNA precursors or pre-miRNA, nor does it compete with and bind to Dicer. It can also be referred to as an expression vector for shRNA or miRNA without inhibiting the function of mature miRNA (see FIG. 12).
  • the Ad genome capsid protein refers to a surface protein of the viral genome, and specifically includes fiber, hexon, penton base, pIX (protein IX) and the like.
  • the fiber protein is composed of a knob, a shaft, and a tail, and is also called a fiber knob, a fiber shaft, and a fiber tail.
  • the Ad vector plasmid of the present invention refers to a plasmid that encodes the Ad vector genome, and may be any plasmid that encodes the genome of the Ad vector having the above-described configuration and can function as an Ad vector plasmid. .
  • a part of the DNA sequence encoding for example, fiber, hexon, penton base, pIX, etc. may be deleted from the Ad genome.
  • a part of the DNA sequence encoding the knob, shaft, tail, etc. may be deleted from the fiber protein.
  • An Ad vector can be prepared by introducing an Ad vector genomic DNA sequence into a specific cell to form Ad vector particles.
  • the present invention also extends to a method for producing the Ad vector of the present invention.
  • Conventional Ad vectors can be prepared using cells that constantly express the E1 gene product of Ad, such as HEK293 cells.
  • HEK293 cells are a cell line established by transforming human fetal kidney cells with the E1 gene of human type 5 Ad, and constantly express the E1 gene product.
  • the Ad vector of the present invention that is, the Ad vector particle of the present invention, cannot be obtained by transfecting the conventionally used HEK293 cells with the Ad vector plasmid of the present invention.
  • a cell for producing the Ad vector of the present invention is a cell capable of inducing the expression of Ad VA-RNAs in a cell that constantly expresses the Ad E1 gene product. It is necessary to be.
  • VA-RNAs are VA-RNA I and / or VA-RNA II.
  • VA-RNA I can be constitutively expressed for Ad VA-RNA I and / or VA-RNA II against cells that constitutively express Ad E1 gene product.
  • a cell line established by transforming part or all of the gene and / or VA-RNA II gene is used. Examples of cells that constantly express the E1 gene product of Ad include HEK293 cells.
  • the present invention also extends to Ad vector production / amplification cells for producing the Ad vector of the present invention.
  • Ad is human type 5 Ad
  • any one selected from the region of the DNA sequence encoding VA-RNA I for example, among the DNA sequences encoding VA-RNA I in human type 5 Ad
  • the human 10620-1079 (SEQ ID NO: 2) of type 5 Ad genome may be introduced.
  • / or any one selected from a region of a DNA sequence encoding VA-RNA II for example, among DNA sequences encoding VA-RNA II in human type 5 Ad, 10876 to 11038 of the human type 5 Ad genome. (SEQ ID NO: 3) may be introduced.
  • the transformed cell can be used as the Ad vector production / amplification cell of the present invention.
  • the method for introducing each sequence for preparing the Ad vector production / amplification cell of the present invention can be a method known per se.
  • a desired viral vector genome such as a lentiviral vector genome can be prepared by incorporating a necessary sequence into a vector and incorporating it into a cell that constantly expresses the E1 gene product of Ad.
  • the present invention also extends to an RNAi induction method characterized by using the above-mentioned Ad vector.
  • shRNA or miRNA By inserting shRNA or miRNA into the Ad vector of the present invention, the function of siRNA or mature miRNA can be exhibited.
  • inducing RNAi RNA interference
  • RNAi refers to suppression of gene expression by siRNA or mature miRNA.
  • the present invention also extends to a kit comprising an Ad vector plasmid for producing the Ad vector of the present invention and the above-described Ad vector production / amplification cells.
  • the Ad vector of the present invention can be easily prepared.
  • the Ad vector of the present invention can be easily amplified, and the Ad vector of the present invention can be used effectively. .
  • the present invention may be a kit including the Ad vector of the present invention and the above-described Ad vector production / amplification cells.
  • the kit By using the kit, the Ad vector of the present invention can be easily amplified, and the Ad vector of the present invention can be used effectively.
  • Ad ⁇ VR vector preparation of an Ad vector that does not express VA-RNAs (Ad ⁇ VR vector)
  • Ad ⁇ VR vector preparation of an Ad vector that does not express VA-RNAs
  • VA-RNAs in this example is used to mean “VA-RNA I and VA-RNA II”.
  • the vector of the present invention is referred to as “Ad ⁇ VR vector”
  • the conventional Ad vector is referred to as “FG-Ad vector”.
  • Each prepared vector has a structure including a gene of GFP, which is a fluorescent protein, and the structure of each vector is shown in FIG.
  • the Ad ⁇ VR vector plasmid lacking the E1 region (342-3523) and the 10667-10703 region and the 10925-10944 region of the genome. was prepared (FIG. 2). Specifically, based on the vector plasmid pAdHM4 capable of inserting a foreign gene into the E1 deletion region, a DNA sequence comprising a VA-RNAs coding sequence in which a part of the internal promoter region of VA-RNA-I and VA-RNA II is deleted and PAdHM4 linearized in the vicinity of the VA-RNAs coding sequence was co-introduced into E. coli BJ5183 strain (Qbiogene) by electroporation to produce homologous recombination to obtain pAdHM4 ⁇ VR.
  • a shuttle plasmid containing a cytomegalovirus (CMV) promoter and a GFP gene as a foreign gene, ie, pHMCMV-GFP was constructed according to a conventional method.
  • Each gene expression shuttle plasmid was cleaved with I-CeuI / PI-SceI and ligated with pAdHM4 ⁇ VR cleaved with I-CeuI / PI-SceI to construct a vector plasmid, ie, pAdHM4 ⁇ VR-CMV GFP.
  • Ad ⁇ VR vector plasmid the vector plasmid prepared in this example
  • FG-Ad vector plasmid a plasmid for preparing an FG-Ad vector.
  • Ad ⁇ VR vector plasmid constructed in 1) above was linearized by cutting with the restriction enzyme PacI present at the end of the viral genome, and transferred to HEK293 cells using the transfection reagent SuperFect TM (Qiagen). Transfected.
  • FG-Ad vector plasmid was transfected by the same technique. After culturing for about 10 days, amplification of the Ad vector was confirmed, but it was confirmed that the Ad ⁇ VR vector was not amplified in HEK293 cells (FIG. 3).
  • Example 2 Construction of Ad ⁇ VR vector production / amplification cells (VR293 cells)
  • amplification was almost achieved in HEK293 cells capable of amplifying conventional FG-Ad vectors. Since it was confirmed that the cells did not, an attempt was made to produce a cell capable of constructing an Ad ⁇ VR vector.
  • a VA-RNA I expression control lentiviral vector (LV-H1tetO-VRI-ERP) containing a cassette that expresses VA-RNA I under the control of the tetracycline-dependent H1 promoter was prepared, introduced into HEK293 cells, and doxycycline ( VR293 cells capable of inducing expression with doxycycline (DOX) were prepared.
  • VA-RNA I in VR293 cells was confirmed by Northern blotting (FIG. 4).
  • the Ad ⁇ VR vector plasmid constructed in Example 1 above was transfected into VR293 cells by the same method as in Example 1, and doxycycline was added to induce the expression of VA-RNA I. It was confirmed that the VR vector was amplified (FIG. 5).
  • Example 1 Confirmation of Ad ⁇ VR Vector Amplification Regarding the Ad ⁇ VR vector amplified in VR293 cells according to Example 2 and the FG-Ad vector amplified in HEK293 cells, the vicinity of the DNA sequence encoding VA-RNAs Amplified by PCR.
  • the amplified product by PCR was treated with the restriction enzyme RsrII, and Ad ⁇ VR vector and FG-Ad vector were confirmed.
  • the Ad ⁇ VR vector prepared in Example 1 lacks a part of the recognition site for the restriction enzyme RsrII in the VA-RNA II gene. Not digested.
  • FIG. 6 shows the results of electrophoresis of each amplified vector using a 2% agarose gel.
  • the Ad ⁇ VR vector did not change in molecular weight even after treatment with the restriction enzyme RsrII, and it was confirmed that the Ad ⁇ VR vector was amplified in VR293 cells.
  • VA-RNA I and VA-RNA II Confirmation of expression of VA-RNAs (VA-RNA I and VA-RNA II) after transduction
  • 0.1 MOI was transduced into HEK293 cells with Ad ⁇ VR vector or FG-Ad vector and cultured for 24 hours
  • the expression of VA-RNA I and VA-RNA II was confirmed.
  • the expression of VA-RNA I and VA-RNA II was confirmed by Northern blotting.
  • expression of VA-RNA I and VA-RNA II was not observed in cells transduced with Ad ⁇ VR vector (FIG. 7).
  • Amplification of the Ad vector genome independent of the E1 gene may increase the expression level of the therapeutic gene, but also increases the expression level of viral proteins that adversely affect the cells. As a result, some cells cause CPE, which triggers inflammation and immune response, and in many cases, amplification of the Ad vector genome independent of the E1 gene is not desired.
  • the Ad ⁇ VR vector of the present invention does not undergo E1 gene-independent genome amplification, and the Ad vector genome does not replicate. As a result, generation of CPE and presentation of viral antigens are suppressed, resulting in inflammation and unnecessary immune responses. Is suppressed.
  • the expression level of the therapeutic gene can be freely controlled by selecting a promoter for controlling the expression of the foreign gene mounted on the foreign gene expression cassette.
  • VA-RNAs may inhibit RNAi induction by shRNA.
  • shRNA In the suppression of the target gene by shRNA, first, the transcribed shRNA is transported to the cytoplasm by Exportin5 and cleaved into siRNA by Dicer. Then, it is taken up by RISC and suppresses the target gene.
  • RISC RNAi induction by Dicer
  • a part of VA-RNAs is also transported to the cytoplasm by Exportin5 like shRNA, and is taken up by RISC after cleavage by Dicer (see FIG. 12). In these processes, it has been reported that VA-RNAs competitively inhibit shRNA knockdown.
  • the Ad vector plasmid and Ad vector were prepared as follows. That is, shLuc expression plasmid pHM5-U6-shLuc (Mizuguchi, et al. Hum Gene Ther., 2007, 18 (1), p.740-80) was treated with SmaI and ligated with an XbaI linker. By ligating a DNA fragment obtained by treating this with XbaI / SphI and a DNA fragment obtained by treating GFP expression plasmid pHM18-CG (Non-patent Document 7) with XbaI / SphI, plasmid pHM18-U6-shLuc-CG was obtained. Obtained.
  • the obtained plasmid pHM18-U6-shLuc-CG was treated with SphI / KpnI-treated DNA fragment and pHM5 (Mizuguchi, et al. Hum Gene Ther., 1999, 10 (12), p.2013-7).
  • the plasmid pHM5-U6-shLuc-CG was obtained by ligating the DNA fragment treated with the enzyme.
  • the obtained plasmid pHM5-U6-shLuc-CG was ligated with the DNA fragment treated with I-CeuI / PI-SceI, and the DNA fragment treated with I-CeuI / PI-SceI with pAdHM4 and pAd ⁇ VR to obtain the plasmid ⁇ pAdHM4 -U6-shLuc-CG "and" pAd ⁇ VR-U6-shLuc-CG "were obtained (see FIG. 13).
  • the Ad ⁇ VR vector plasmid pAd ⁇ VR-shLuc-CG was linearized by cutting with restriction enzyme PacI is present recognition sites at both ends of the Ad genome, the VR293 cells using Lipofectamine TM 2000 (Invitrogen) Transfected and prepared.
  • VR293 cells were prepared by the method described in Example 2. After transfection, the cells were cultured in a normal medium for 4 days, and then further cultured for 9 days in a medium containing Dox (50 ng / ml) as a transduction reagent. Thereafter, the cells were collected, and the cells were destroyed by freezing and thawing.
  • Luc expressing cells were prepared as follows. Specifically, SK HEP-1 cells were seeded in a 12-well plate at 1 ⁇ 10 5 cells / well, and the next day, a culture supernatant 1 containing a luciferase-expressing lentiviral vector (LV-EVLuP; also equipped with a Venus gene encoding a fluorescent protein) ml was allowed to act. After culturing for 2 days, the virus infection efficiency was confirmed by flow cytometry (FACS). After culturing and scaling up, luciferase-expressing SK HEP-1 cells (SK HEP-1-Luc cells) were obtained by sorting Venus positive cells with FACSAria TM (Becton Dickinson Co., Ltd.).
  • LV-EVLuP luciferase-expressing lentiviral vector
  • SK HEP-1-Luc cells were seeded at 1 ⁇ 10 4 cells / wel in a 96-well black plate, and the following day, each Ad vector was allowed to act at 1, 3 and 10 MOI at 37 ° C. for 90 minutes. After culturing for 36 hours, luciferase activity was measured using Pica Gene LT 2.0 (Toyo Ink).
  • the Ad vector in which the VA-RNAs gene of the present invention is modified suppresses Ad vector genome amplification in cells except for special cells, and effectively puts the desired gene into the cells. Can be introduced. Since conventional Ad vectors lack the E1 region, they are theoretically amplified only in special cells that express the E1 gene product. However, it is known that the vector genome is amplified independently of the E1 gene in certain cells. In addition, it is known that transcription of the viral protein coding gene remaining in the vector genome occurs independently of the E1 gene. Amplification of the Ad vector genome independent of the E1 gene also increases the amount of viral protein expression that adversely affects cells. As a result, some cells may experience CPE, which can trigger inflammation and immune responses.
  • the Ad vector genome does not replicate in the Ad vector in which the VA-RNAs gene of the present invention is modified, the generation of CPE and the presentation of viral antigens are suppressed as a result, and inflammation and unnecessary immune reactions are suppressed. Therefore, the Ad vector in which the VA-RNAs gene of the present invention is modified does not cause E1 gene-independent genome amplification, and the safety is drastically increased while maintaining the characteristics of Ad vector with high gene transfer efficiency. It can be called a vector (see FIGS. 9 to 11). In addition, since the possibility that the transgenic cells are eliminated due to the attenuation of the immune response is reduced, the gene expression period can be expected to be extended.
  • the Ad vector deficient in the VA-RNAs of the invention RNAi is effectively induced without suppressing the generation of mature miRNA or siRNA. Based on the above, the Ad vector deficient in VA-RNAs of the present invention is useful as a gene introduction tool in improving the safety of gene therapy and life science research, and develops an shRNA expression Ad vector with a high knockdown effect of the target gene be able to.
  • an Ad vector in which the VA-RNAs gene of the present invention is modified can be prepared from an Ad vector genomic DNA sequence in which the VA-RNAs gene is modified.
  • Ad vectors can be amplified.

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Abstract

La présente invention concerne un vecteur Ad très sûr présentant une efficacité de transfert génique équivalente ou supérieure à celle de vecteurs Ad classiques; à savoir, un vecteur Ad apte à être utilisé efficacement et de façon sûre en thérapie génique et en recherche en science de la vie. L'invention concerne en outre des cellules amplificatrices/productrices de vecteurs Ad aptes à amplifier le vecteur Ad. Selon ce vecteur Ad, un gène VA-ARN (VA-ARN I et/ou VA-ARN II) est modifié dans le génome du vecteur Ad de sorte que les VA-ARN ne sont pas exprimés. Dans des cellules amplificatrices/productrices de vecteurs Ad, un gène est introduit afin que les VA-ARN du génome Ad soient exprimés dans des cellules exprimant d'une manière constitutive un produit de gène E1 utilisé pour amplifier le vecteur Ad.
PCT/JP2012/055027 2011-03-04 2012-02-29 Vecteur adénoviral dans lequel les va-arn ne sont pas exprimés WO2012121071A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014050927A1 (fr) * 2012-09-26 2014-04-03 国立大学法人 東京大学 Vecteur d'adénovirus à disruption de gène va, et vecteur précurseur permettant de le préparer
JPWO2014050927A1 (ja) * 2012-09-26 2016-08-22 国立大学法人 東京大学 Ｖａ遺伝子破壊アデノウイルスベクターおよびそれを調製するための前駆体ベクター

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