WO2015025940A1 - Nouvel adénovirus et procédé pour favoriser la croissance de celui-ci - Google Patents

Nouvel adénovirus et procédé pour favoriser la croissance de celui-ci Download PDF

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WO2015025940A1
WO2015025940A1 PCT/JP2014/071960 JP2014071960W WO2015025940A1 WO 2015025940 A1 WO2015025940 A1 WO 2015025940A1 JP 2014071960 W JP2014071960 W JP 2014071960W WO 2015025940 A1 WO2015025940 A1 WO 2015025940A1
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mir
dicer
ago2
adenovirus
cells
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水口　裕之
櫻井　文教
充洋 町谷
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国立大学法人大阪大学
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Definitions

  • the present invention relates to an adenovirus (hereinafter referred to as “Ad”) that can effectively propagate in desired cells. Furthermore, it relates to a method for promoting the proliferation of Ad.
  • Ad adenovirus
  • Ad vectors are used for gene transformation research and production of genetically engineered cells for industrial use as gene transfer vectors due to their excellent gene transfer characteristics, and also applicable to various diseases as gene therapy vectors. Is expected.
  • the limited growth type Ad is characterized in that it does not grow on normal cells but grows only on tumor cells or special cells. Infection with Ad (type 2 or type 5) begins when the knob at the end of the fiber protruding from the viral protein shell (capsid) is adsorbed on the cell surface coxsackie-adenovirus receptor (CAR). The penton base at the base of the fiber binds and enters the cell.
  • CAR coxsackie-adenovirus receptor
  • Ad is considered to have a higher efficiency in transferring the viral genome into the nucleus than other DNApox viruses, resulting in higher gene transfer efficiency as a vector.
  • Ad replication begins by first expressing the E1A protein and transactivating the expression of all other early proteins, E1B, E2, E3 and E4. Since the E1 substitution vector generally applied lacks E1A and E1B, HEK293 cells in which the E1 protein is constitutively expressed can replicate almost the same as the wild type. In cells other than HEK293 cells, the vector genome is efficiently transferred into the nucleus according to the previous invasion method, but since no protein expression from all subsequent viruses occurs, it does not replicate as a virus. Such vectors are classified as non-proliferating. On the other hand, attempts have been made to replicate Ad only in tumor cells and kill the tumor cells.
  • Restricted growth type Ad which does not proliferate in normal cells but specifically kills tumor cells by infecting and proliferating in tumor cells, is expected as a next-generation tumor therapeutic agent.
  • E1B mutant viruses that can grow only in p53-deficient cells and E1A are expressed from tumor cell-specific promoters, and restricted-proliferation type Ad has been developed that replicates only in tumor cells.
  • restricted-proliferation type Ad has been developed that replicates only in tumor cells.
  • a technology has been developed in which a reporter gene such as GFP is mounted on a restricted growth type Ad, and is replicated only in tumor cells so that GFP is produced and fluorescent light is emitted to detect tumor cells.
  • the conventional limited growth type Ad has a problem that a sufficient therapeutic effect cannot be obtained due to a slow growth rate in tumor cells.
  • Ad production packaging cells for producing restricted-proliferation type Ad, non-proliferation type Ad or other Ad used for gene transfer vectors, gene therapy vectors, tumor therapeutic agents, tumor detection agents, and other uses
  • Ad production packaging cells for producing restricted-proliferation type Ad, non-proliferation type Ad or other Ad used for gene transfer vectors, gene therapy vectors, tumor therapeutic agents, tumor detection agents, and other uses
  • the growth rate of Ad is slow, a sufficient amount of Ad cannot be obtained effectively.
  • VA-RNA which is a small molecule RNA, can be cited as a viral factor that promotes Ad growth.
  • VA-RNA includes VA-RNA I and VA-RNA II, both of which are untranslated RNA polymerase III transcripts (single-stranded RNA) of about 160 bases in length, especially VA-RNA I, which has a large amount of transcription Reaches ⁇ 10 8 molecules per cell during Ad replication.
  • VA-RNA I plays a central role in inhibiting the 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 has been reported that PKR is activated and phosphorylation of the translation initiation factor eIF-2 ⁇ is induced, resulting in inhibition of translation of viral mRNA (Non-patent Documents 1 and 2). That is, it is considered that VA-RNA promotes the translation of the viral gene by inhibiting PKR and thus promotes the growth of the virus. It has also been reported that VA-RNA inhibits ADAR (RNA-specific adenosine deaminase) (Non-patent Document 3).
  • ADAR RNA-specific adenosine deaminase
  • VA-RNA is transcribed in the nucleus, it is exported to the nucleus, that is, to the cytoplasm by Exportin-5 (XPO5), which is a nuclear export protein, and after being cleaved by Dicer, forms mivaRNA (viral miRNA),
  • XPO5 Exportin-5
  • RISC RNA-induced silencing complex
  • Ago Argonaute protein
  • Non-patent Document 6 the growth of Ad is suppressed by introducing mivaRNA antisense oligonucleotides into cells.
  • An object of the present invention is to provide Ad that can effectively proliferate in desired cells such as target tumor cells. Another object of the present invention is to provide a method for promoting the growth of Ad, in which Ad is effectively propagated in desired cells. Furthermore, an object of the present invention is to provide an Ad-producing packaging cell that can effectively proliferate Ad.
  • the present inventors focused on the relationship between Ad-derived mivaRNA and virus growth in the Ad genome, and as a result of intensive studies, conventionally, Ad-derived mivaRNA has suppressed gene expression in infected cells. Despite being thought to promote virus growth (Non-Patent Documents 5 and 6), surprisingly, Ad effectively proliferated in cells by suppressing the function of Ad-derived mivaRNA. The inventors have found that the problems can be solved and completed the present invention. Moreover, it discovered that the viral growth promotion effect of VA-RNA can be acquired by suppressing the cutting
  • this invention consists of the following. 1. A method for promoting the growth of adenovirus, comprising suppressing the function of adenovirus-derived mivaRNA. 2. A method for promoting the growth of adenovirus, wherein the method promotes the growth of adenovirus by inhibiting miR-27a and / or miR-27b. 3. 3. The method of promoting the growth of adenovirus according to item 1 or 2, wherein suppressing mivaRNA function or inhibiting miR-27a and / or miR-27b inhibits at least one of Dicer and Ago2. 4). 4.
  • 3. The method for promoting the growth of an adenovirus according to item 2 wherein the inhibition of miR-27a or miR-27b is inhibition by a decoy nucleic acid for miR-27a or miR-27b, or an inhibitor for miR-27a or miR-27b.
  • An adenovirus comprising an siRNA expression cassette for Dicer or Ago2 or a decoy nucleic acid expression cassette for miR-27a or miR-27b in the adenovirus genome.
  • the siRNA for Dicer is RNA containing the oligonucleotide shown in 1) or 2) below
  • the siRNA for Ago2 is RNA containing the oligonucleotide shown in 3) or 4) below
  • the decoy nucleic acid for miR-27a Is an RNA containing the oligonucleotide shown in 5) below
  • the decoy nucleic acid against miR-27b is an RNA containing the oligonucleotide shown in 6) below; 1) GAAUCAGCCUCGCAACAAA (SEQ ID NO: 1) 2) UUUGUUGCGAGGCUGAUUC (SEQ ID NO: 2) 3) GCACGGAAGUCCAUCUGAA (SEQ ID NO: 3) 4) UUCAGAUGGACUUCCGU
  • adenovirus according to item 9 or 10 above, wherein the adenovirus is a restricted propagation adenovirus.
  • An antitumor agent or tumor detection agent comprising the restricted-proliferative adenovirus according to item 11 as an active ingredient.
  • 13 In the genome of adenovirus-producing packaging cells, an adenovirus is produced, characterized in that at least an expression cassette of siRNA for Dicer or Ago2 or an expression cassette of miR-27a or miR-27b decoy nucleic acid is introduced Packaging cells to do.
  • the siRNA for Dicer is RNA containing the oligonucleotide shown in 1) or 2) below, and the siRNA for Ago2 is RNA containing the oligonucleotide shown in 3) or 4) below, and the decoy nucleic acid for miR-27a Is a RNA containing the oligonucleotide shown in 5) below, and the decoy nucleic acid against miR-27b is an RNA containing the oligonucleotide shown in 6) below: 1) GAAUCAGCCUCGCAACAAA (SEQ ID NO: 1) 2) UUUGUUGCGAGGCUGAUUC (SEQ ID NO: 2) 3) GCACGGAAGUCCAUCUGAA (SEQ ID NO: 3) 4) UUCAGAUGGACUUCCGUGC (SEQ ID NO: 4) 5) GCGGAACUUAGAUCUCCACUGUGAA (SEQ ID NO: 10) 6) GCAGAACUUAGAUCUCCACUGUGAA (SEQ ID NO
  • a method for treating a malignant tumor comprising administering the antitumor agent according to 1.
  • B. A method for producing Ad, comprising a step of suppressing the function of Ad-derived mivaRNA in a system that produces Ad using an Ad genome and an Ad-producing packaging cell.
  • C. A method for producing Ad, comprising a step of inhibiting miR-27a and / or miR-27b in a system that produces Ad using an Ad genome and an Ad-producing packaging cell.
  • E The method for producing Ad according to item D, wherein the inhibition of Dicer or Ago2 is inhibition of Dicer or Ago2 by RNA interference.
  • F. Inhibition of Dicer or Ago2 by RNA interference is due to the expression of siRNA against Dicer mounted on Ad or the expression of siRNA against Ago2, or the expression of siRNA against Dicer or the siRNA against Ago2 introduced into Ad production packaging cells.
  • G The production method according to item D, wherein the inhibition of Dicer or Ago2 is inhibition by knockout of the Dicer gene or Ago2 gene in Ad production packaging cells.
  • the production method according to item D wherein the inhibition of Dicer or Ago2 is inhibition by an antibody of Dicer or Ago2, or inhibition by an inhibitor of Dicer or Ago2.
  • the production method according to item C wherein the inhibition of miR-27a or miR-27b is inhibition by a decoy nucleic acid for miR-27a or miR-27b, or an inhibitor for miR-27a or miR-27b.
  • the Ad of the present invention can promote the proliferation of Ad more effectively than the conventional Ad. According to the antitumor agent containing Ad of the present invention as an active ingredient, proliferation is effectively promoted in the target tumor cells, and an antitumor effect is exhibited.
  • Ad can be effectively proliferated in the desired cells, such as target tumor cells and Ad proliferation cells, it will effectively demonstrate anti-malignant tumor effects such as tumor lysis, tumor detection effects, and efficient Ad production effects Yes.
  • Ad-shDicer a figure which shows typically the structure of the genome of Ad (henceforth Ad produced in Example 1 is called "Ad-shDicer") carrying shRNA (short hairpin RNA) with respect to Dicer.
  • Ad-shDicer a figure which shows the result of having confirmed the genome growth ability of Ad-shDicer in various human cancer cells.
  • Example 1-1 It is a figure which shows the result of having confirmed the cell survival rate when making Ad-shDicer act on various human cancer cells.
  • Example 1-2 It is a figure which shows the result of having confirmed the cell survival rate when making Ad-shDicer act on various human normal cells.
  • Example 1-2 It is a figure which shows the relative value by measuring the amount of Dicer® mRNA when Ad-shDicer is allowed to act on various cancer cells.
  • Example 1-3 It is a conceptual diagram which shows the process in which Ad-derived VA-RNA is cleaved by Dicer and forms an Ago2 protein and RISC complex.
  • Example 1-4 It is a figure which shows the result of having confirmed the viral genome amount when adding wild type Ad to the said various cells made to interfere with RNA by siRNA introduction
  • Example 1-4 It is a figure which shows the result of having confirmed the influence which it has on the production of Ad origin VA-RNA I or VA-RNA II when introducing siRNA with respect to Ago2 or Dicer into various cancer cells by electrophoresis.
  • Example 1-4 It is a figure which shows the result of having confirmed the relative expression level of Ad origin mivaRNA (TM) I or mivaRNA (TM) II when siRNA with respect to Ago2 or Dicer is introduce
  • TM Ad origin mivaRNA
  • TM mivaRNA
  • Example 1-5 It is a figure which shows the result of having confirmed the viral genome amount when adding wild-type Ad to each cell when Ago2 or Dicer is overexpressed in various cancer cells.
  • Comparative Example 1 It is a figure which shows the result of having confirmed the cell killing effect with respect to the cancer cell of Ad-shDicer.
  • Example 2 It is a figure which shows the result of having confirmed the anticancer effect
  • Example 3 It is a figure which shows the result of having confirmed the expression level of miR-27a, b in a Dicer knockdown cell.
  • Example 4 The genome structure of an Ad carrying a tough decoy RNA against miR-27a or miR-27b (hereinafter, the Ad produced in Example 4 is referred to as “Oncolytic Ad-TuD-27a” or “Oncolytic Ad-TuD-27b”). It is a figure shown typically.
  • Example 4 It is a figure which shows the result of having confirmed the cancer cell survival rate when making Oncolytic Ad-TuD-27a or Oncolytic Ad-TuD-27b act.
  • Example 4-1 It is a figure as a result of confirming Ad genome growth ability in a cancer cell by Oncolytic®Ad-TuD-27a or Oncolytic®Ad-TuD-27b.
  • Example 4-2 It is a figure as a result of confirming Ad genome growth ability in a cancer cell by Oncolytic®Ad-TuD-27a or Oncolytic®Ad-TuD-27b.
  • Ad means an adenovirus
  • Ad genome means genetic information possessed by Ad, that is, a DNA sequence contained in Ad.
  • the present invention relates to a method for promoting the proliferation of Ad, including suppressing the function of Ad-derived mivaRNA.
  • “suppressing the function of mivaRNA” means “inhibition of mivaRNA production” or “inhibition of mivaRNA activity”. Specifically, it means “inhibition of mivaRNA production by inhibiting cleavage of VA-RNA” or “inhibition of mivaRNA activity by inhibiting RISC formation of mivaRNA and Ago2.” Such “suppressing the function of mivaRNA” is achieved by inhibiting at least one of Dicer and Ago2. In addition, by inhibiting Dicer, “inhibiting cleavage of VA-RNA”, it is possible to obtain a virus growth promoting effect of VA-RNA.
  • the present invention also relates to a method for promoting the proliferation of Ad by inhibiting miR-27a and / or miR-27b.
  • inhibition of miR-27a and / or miR-27b is achieved by inhibiting at least one of Dicer and Ago2.
  • Other methods include, for example, inhibition by mico-27a or miR-27b described later with a decoy nucleic acid, inhibition with an inhibitor against miR-27a or miR-27b, inhibition by miR-27a or miR-27b knockout, etc. .
  • the method for inhibiting Dicer or Ago2 is not particularly limited, but for example, inhibition by RNA interference with Dicer or Ago2, inhibition by Dicer gene or Ago2 gene knockout, or inhibition by using antibodies or inhibitors against Dicer or Ago2 etc. Can be mentioned. Inhibition by RNA interference and inhibition by gene knockout are preferred, and inhibition by RNA interference is most preferred. Inhibition by RNA interference with Dicer or Ago2 can be achieved by allowing the Ad genome or Ad producing cells to act with siRNA against Dicer or Ago2. In order to cause siRNA to act on Dicer or Ago2, an siRNA expression cassette for Dicer or Ago2 can be mounted on the Ad genome or introduced into an Ad-producing cell. Moreover, siRNA for Dicer or Ago2 may be added to the culture system of Ad-producing cells.
  • the siRNA for Dicer or Ago2 may be any siRNA having an RNA interference function for Dicer or Ago2, and is not particularly limited and may be any siRNA known per se or developed in the future.
  • Examples of siRNA and shRNA having an RNA interference function for Dicer known per se include shRNAs shown in the following papers. Inhibition of Dicer or Ago2 by RNA interference and inhibition of Dicer gene or Ago2 gene by knockout are known methods such as NucleicNAcids Research, (2006) Vol. 34, No. 17 4801-4815 (doi: 10.1093 / nar / gkl646), Cell 141, 1195-1207, June 25 (2010), Molecular Therapy vol. 17 no. 4, 725-732 apr.
  • siRNA against Dicer examples include oligonucleotides having the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 below. ⁇ GAAUCAGCCUCGCAACAAA (SEQ ID NO: 1) ⁇ UUUGUUGCGAGGCUGAUUC (SEQ ID NO: 2)
  • an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5 is used as a vector based on the sequence of shRNA that is the precursor of siRNA. It can be mounted and used.
  • the base sequence shown in SEQ ID NO: 6 or 7 is the base sequence of the underlined part in SEQ ID NO: 5, and this part is considered to be the most important part in RNA interference action.
  • GATCCC GAATCAGCCTCGCAACAAA TTCAAGAGA TTTGTTGCGAGGCTGATTC TTTTTGGAAAT (SEQ ID NO: 5)
  • GAATCAGCCTCGCAACAAA GAATCAGCCTCGCAACAAA
  • SEQ ID NO: 7 ⁇ TTTGTTGCGAGGCTGATTC (SEQ ID NO: 7)
  • siRNA against Ago2 include oligonucleotides comprising the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 below.
  • GCACGGAAGUCCAUCUGAA SEQ ID NO: 3
  • UUCAGAUGGACUUCCGUGC SEQ ID NO: 4
  • siRNA against Dicer or Ago2 when RNA interference is used to inhibit Dicer or Ago2, it can be achieved by inhibition by expression of siRNA against Dicer or Ago2 mounted on Ad. Similarly, it can be achieved by expression of siRNA against Dicer or Ago2 introduced into packaging cells for producing Ad. Specific examples of siRNA against Dicer or Ago2 are as described above.
  • the “expression of siRNA against Dicer or Ago2” may be an siRNA expression cassette containing siRNA against Dicer or Ago2.
  • siRNA expression cassette containing “siRNA against Dicer or Ago2” may be, for example, an siRNA expression cassette containing “siRNA against Dicer or Ago2,” or an siRNA expression cassette containing “shRNA against Dicer or Ago2.” There may be. Since shRNA is a precursor of siRNA, it is also included in “expression of siRNA” in this specification that siRNA is produced after shRNA is expressed.
  • Dicer or Ago2 when Dicer or Ago2 is inhibited using a Dicer or Ago2 antibody, or a Dicer or Ago2 inhibitor, a known antibody or inhibitor (decoy nucleic acid or aptamer nucleic acid is used). Can be used).
  • Dicer or Ago2 of Ad proliferation cells By inhibiting Dicer or Ago2 of Ad proliferation cells with the antibody or the inhibitor, Ad proliferation in the Ad proliferation cells can be promoted.
  • a decoy nucleic acid against miR-27a or miR-27b can act on Ad to inhibit miR-27a or miR-27b.
  • a decoy nucleic acid expression cassette for miR-27a or miR-27b can be mounted on the Ad genome or introduced into Ad-producing cells.
  • an antisense oligonucleotide against miR-27a or miR-27b or a reagent commercially available as an inhibitor of miR-27a or miR-27b, such as miRVana miRNA inhibitor (Ambion) may be used.
  • the decoy nucleic acid for miR-27a may be a sequence containing at least one or more of the base sequence shown in SEQ ID NO: 10.
  • Specific examples of the decoy nucleic acid for miR-27a include an oligonucleotide having the base sequence shown in SEQ ID NO: 12 below.
  • the underlined part of SEQ ID NO: 12 is an antisense sequence for miR-27a, which corresponds to the base sequence shown in SEQ ID NO: 10.
  • the decoy nucleic acid for miR-27b may be a sequence containing at least one or more of the base sequence shown in SEQ ID NO: 11.
  • a specific example of a decoy nucleic acid for miR-27b includes an oligonucleotide having the base sequence shown in SEQ ID NO: 13 below.
  • the underlined part of SEQ ID NO: 13 is an antisense sequence for miR-27b and corresponds to the base sequence shown in SEQ ID NO: 11.
  • an oligonucleotide necessary as appropriate may be included.
  • GCGCAACUUAGAUCUCCACUGUGAA (SEQ ID NO: 10) GCAGAACUUAGAUCUCCACUGUGAA (SEQ ID NO: 11) ⁇ GACGGCGCUAGGAUCAUCAAC GCGGAACUUAGAUCUCCACUGUGAA CAAGUAUUCUGGUCACAGAAUACAAC GCGGAACUUAGAUCUCCACUGUGAA CAAGAUGAUCCUAGCGCCGUCUU (SEQ ID NO: 12) ⁇ GACGGCGCUAGGAUCAUCAAC GCAGAACUUAGAUCUCCACUGUGAA CAAGUAUUCUGGUCACAGAAUACAAC GCAGAACUUAGAUCUCCACUGUGAA CAAGAUGAUCCUAGCGCCGUCUU (SEQ ID NO: 13)
  • Examples of the Ad whose proliferation is promoted in the “method for promoting the proliferation of Ad” of the present invention include an Ad that can be replicated only in a specific cell type, that is, a restricted proliferation Ad and a non-proliferation Ad vector. Particularly preferred is restricted growth type Ad.
  • restricted growth type Ad As shown in the background art section of this specification, a restricted growth type Ad that does not grow in normal cells but specifically infects and proliferates and kills tumor cells or detects tumor cells is a next generation tumor. Although it has been expected as a therapeutic drug and a tumor detection drug, the conventional limited growth type Ad has a problem that a sufficient therapeutic effect cannot be obtained due to a slow growth rate in tumor cells.
  • Ad growth promotion method an antitumor agent or tumor cell that causes Ad to replicate only in tumor cells and kills tumor cells is detected. It can be effectively used as a detection agent.
  • Restricted growth type Ad is an Ad that occurs only in tumor cells, for example, and can be exemplified by those reported in NATURE MEDICINE, 7, 781-787 (2001).
  • Ad carrying a promoter that controls gene expression in a tumor cell-specific manner can be mentioned. As long as it is a promoter capable of exhibiting such a function, either a promoter known per se or a promoter developed in the future may be used.
  • Examples include hTERT promoter, IAI.3B promoter, midkine promoter, ⁇ -HCG promoter, SCCA1 promoter, cox-2 promoter, PSA promoter, or other tumor-specific promoters.
  • a suitable promoter that can correspond to the virus used for expression of the target gene, in addition to a tumor-specific promoter, for example, CMV promoter, SV40 late promoter, MMTV LTR promoter, RSV LTR promoter, SR ⁇ promoter, etc. may be used. it can.
  • the “Ad” that promotes proliferation of the present invention must be devised to suppress the function of Ad-derived mivaRNA or to inhibit miR-27a and / or miR-27b in the Ad genome.
  • expression cassettes such as decoy nucleic acids and aptamer nucleic acids for Dicer or Ago2
  • expression cassettes such as antisense oligonucleotides for Dicer or Ago2 genes may be mounted.
  • expression cassettes such as decoy nucleic acids and aptamer nucleic acids for miR-27a or miR-27b, antisense oligonucleotides for miR-27a or miR-27b, etc.
  • the expression cassette may be mounted.
  • the siRNA or shRNA for Dicer and Ago2 or the decoy nucleic acid for miR-27a or miR-27b is as described above.
  • Ad is typically a human type 2, type 5, type 11, type 35 Ad, a non-human type host Ad, mouse Ad, dog Ad, sheep Ad and bird Ad and so on.
  • the E1 region-deficient Ad refers to an Ad that is functionally deficient in the E1 gene region so that the E1 protein is not expressed in the Ad genome.
  • 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 functional deletion of the E1 gene region means, for example, that the E1 protein that functions in the host cell is not expressed, and it is not necessary to delete all of the genomic region encoding the E1 protein.
  • the Ad that can be used for the Ad of the present invention may be, for example, a 5-type Ad that functionally lacks the E3 gene region in addition to the E1 region.
  • the E3 region of the type 5 Ad genome (GenBank Accession number: M73260, M29978) refers to a site corresponding to the 28133-30308th or 27865-30309th.
  • Ad can be promoted by applying the “method for promoting the proliferation of Ad” of the present invention to at least either “Ad” or “Ad proliferation cell”.
  • the Ad in this case is not limited to the restricted growth type Ad, and may be a non-growth type Ad. Regardless of the restricted growth type Ad or the non-growth type Ad, Ad can be effectively utilized if virus growth can be promoted in vitro.
  • Examples of the “Ad proliferation cell” for producing Ad by effectively promoting proliferation include “Ad production packaging cell”.
  • Ad production packaging cell is not particularly limited as long as it is a cell capable of producing Ad.
  • examples of cells that can be used as packaging cells for producing Ad include HEK293 cells in addition to various tumor cells.
  • Ad production packaging cells to effectively promote growth of Ad are designed to suppress the function of Ad-derived mivaRNA or to inhibit miR-27a and / or miR-27b It must be a cell.
  • An expression cassette such as a decoy nucleic acid or aptamer nucleic acid for Dicer or Ago2 or an antisense cassette for a Dicer or Ago2 gene may be introduced.
  • expression cassettes such as decoy nucleic acids and aptamer nucleic acids for miR-27a or miR-27b, antisense oligonucleotides for miR-27a or miR-27b, etc.
  • the expression cassette may be introduced.
  • the siRNA or shRNA for Dicer and Ago2 or the decoy nucleic acid for miR-27a or miR-27b is as described above.
  • the present invention extends to an antitumor agent or a tumor detection agent containing the Ad of the present invention as an active ingredient.
  • the antitumor agent of the present invention includes tablets, capsules, powders, granules, pills, liquids, syrups and other oral administration agents, injections, external preparations, suppositories, ophthalmic preparations and the like. Depending on the form, it can be administered orally or parenterally. Preferably, local injection into a tumor, muscle, abdominal cavity, etc., injection into a vein, etc. are exemplified.
  • the antitumor agent or tumor detection agent containing an effective amount of Ad of the present invention is produced according to a conventional method, and generally a physiological saline, a cell culture solution or the like can be used as a diluent. Further, if necessary, bactericides, preservatives, stabilizers, tonicity agents, soothing agents and the like may be added.
  • the antitumor agent of the present invention can be applied to almost all malignant tumors, and the types of cancer to be treated are ovarian cancer, squamous cell carcinoma (cervical cancer, skin cancer, head and neck cancer, esophageal cancer, Lung cancer etc.), digestive organ cancer (colon cancer, pancreatic cancer, liver cancer, gastric cancer etc.), neuroblastoma, brain tumor, breast cancer, testicular cancer, prostate cancer and the like.
  • ovarian cancer squamous cell carcinoma
  • squamous cell carcinoma cervical cancer, skin cancer, head and neck cancer, esophageal cancer, Lung cancer etc.
  • digestive organ cancer colon cancer, pancreatic cancer, liver cancer, gastric cancer etc.
  • neuroblastoma brain tumor, breast cancer, testicular cancer, prostate cancer and the like.
  • the present invention extends to a method for treating malignant tumors. Specifically, it is by administering an effective amount of the antitumor agent of the present invention for the treatment and / or prevention of any of the aforementioned malignant tumors.
  • the antitumor agent of the present invention can be applied to the affected area as it is, or any known method such as intravenous, intramuscular, intraperitoneal or subcutaneous injection, inhalation from the nasal cavity, oral cavity or lung, oral administration, It can also be introduced into a living body (target cell or organ) by intravascular administration using a catheter or the like.
  • a known pharmaceutically acceptable carrier such as an excipient, a bulking agent, a binder or a lubricant, a known additive (buffering agent, isotonicity) Agents, chelating agents, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, etc.).
  • the dose for treatment can be appropriately determined according to the size / type of the tumor, the degree of symptoms, the administration route, the administration subject, the age of the patient, the body weight, and the like.
  • the antitumor agent of the present invention may be administered to a patient several times, or may be divided into multiple courses, and the number of administrations per course, the administration interval, etc. may be arbitrarily set.
  • the amount of the virus of the present invention which is usually an active ingredient, can be about 10 6 to 10 11 PFU (plaque forming units), preferably about 10 9 to 10 11 PFU.
  • the antitumor agent of this invention can also make it easy to infect Ad contained as an active ingredient by suppressing a living body's immunity by using a well-known immunosuppressive agent etc.
  • the antitumor agent of the present invention can be used in combination with at least one anticancer agent selected from the group consisting of known anticancer agents and radiation.
  • Anticancer agents include, but are not limited to: (A) Alkylation activator: This preparation has an action of introducing an alkyl group into a nucleic acid protein of cancer cells to cause cell damage. For example, carbocon, psulfan (mustard drug), nimustine (nitrosoureas) ) Etc.
  • Antimetabolite active agent This preparation has an action of antagonizing an enzyme in the metabolic process and inhibiting cell synthesis. For example, methotrexate (folic acid), mercaptopurine (purine), cytarabine (pyrimidine) System), fluorouracil, tegafur, carmofur and the like.
  • C Antibiotics: Actinomycin D, preomycin, adriamycin, mitomycin C, etc. having anticancer activity.
  • Microtubule inhibitory active agent This preparation acts on microtubules and exhibits an antitumor effect, such as docetaxel, paclitaxel (taxane), vinorelbine, pinklistin, vinblastine (alkaloids), etc.
  • Platinum preparation This preparation has an action of inhibiting DNA synthesis by forming intra-DNA chain or inter-chain bond or DNA protein bond, and examples thereof include cisplatin, arboplatin, nedaplatin and the like.
  • Topoisomerase inhibitory activator Examples include irinotecan (topoisomerase I inhibitor), podophyllotoxin derivative (topoisomerase II inhibitor), which inhibit topoisomerase. Topoisomerase is an enzyme that catalyzes a reaction that temporarily cuts DNA and changes the linking number of DNA strands.
  • the present invention extends to a highly sensitive detection method for malignant tumors. Specifically, for detection of any of the above-mentioned malignant tumors, the target cell, cultured cell, cultured tissue, collected blood or biopsy tissue of the tumor detection agent of the present invention loaded with a reporter gene such as GFP Or by administering to a living body.
  • the tumor detection agent of the present invention can be applied to the affected part of a living body as it is, or any known method, for example, injection such as vein, muscle, intraperitoneal or subcutaneous, inhalation from nasal cavity, oral cavity or lung, oral It can also be introduced into a living body (target cells or organs) by administration, intravascular administration using a catheter or the like.
  • a known pharmaceutically acceptable carrier such as an excipient, a bulking agent, a binder or a lubricant, a known additive (buffering agent, isotonicity) Agents, chelating agents, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, etc.).
  • the amount used to detect malignant tumors is appropriately determined according to the collection conditions, culture conditions, tumor size / type, degree of symptoms, administration route, administration target, animal species, patient age, body weight, etc. be able to.
  • the tumor detection agent of the present invention may be administered to a living body several times, or may be divided into multiple courses, and the number of administrations per course, the administration interval, etc. may be arbitrarily set. It is also possible to collect and inspect blood and biopsy material from a living body. For example, referring to J. Clin. Invest., 119: 3172-3181 (2009) and VNATURE MEDICINE, 12: 1214-12193181 (2006), the amount of use of the tumor detection agent of the present invention can be appropriately determined. .
  • Ad as an active ingredient can be promoted effectively compared with the conventional method.
  • the cells in which the proliferation of Ad of the present invention is promoted include ovarian cancer, squamous cell carcinoma (cervical cancer, skin cancer, head and neck cancer, esophageal cancer, lung cancer, etc.), digestive organ cancer (colon cancer, pancreatic cancer, liver cancer, gastric cancer). Etc.), target tumor cells such as neuroblastoma, brain tumor, breast cancer, testicular cancer, prostate cancer and the like. If Ad can be effectively proliferated in a desired cell such as a target tumor cell, an anti-malignant tumor effect such as tumor lysis or a tumor detection effect can be effectively exhibited. Then, by applying the growth promotion method of the present invention to an in vitro vitro Ad production system using cells, such as a virus production system using HEK293 cells, Ad can be produced more effectively. it can.
  • Example 1 Production of Ad loaded with shRNA against Dicer
  • a cassette loaded with shRNA against Dicer hereinafter referred to as "shDicer” in the Examples and Experimental Examples
  • Ad-shDicer The production of Ad carrying a cassette capable of expressing siRNA will be described.
  • Ad-shDicer The genomic structure of the produced Ad-shDicer is as shown in FIG.
  • the human type 5 Ad genome (GenBank Accession No .: M73260, M29978) lacks the E1 region (342-3523) and E3 region (28133-30818) of the genome. It was constructed based on a vector plasmid that can insert a foreign gene into the E1-deficient region.
  • the hTERT promoter which is a tumor-specific promoter
  • E1A, IRES, E1B, and polyA (pA) signal sequences are ligated and mounted in the E1 deficient region of the vector plasmid pAdHM19 to obtain an oncolytic Ad genome. It was.
  • the shDicer expression cassette was mounted in the E3-deficient region.
  • the sequence corresponding to shDicer in the shDicer expression cassette used in this example consists of the base sequence shown in SEQ ID NO: 5. What is important for exerting the RNA interference function is considered to be the base sequence shown underlined in SEQ ID NO: 5, specifically the base sequence part shown in SEQ ID NO: 6 or SEQ ID NO: 7. (SEQ ID NO: 5) GATCCC GAATCAGCCTCGCAACAAA TTCAAGAGA TTTGTTGCGAGGCTGATTC TTTTTGGAAAT (SEQ ID NO: 6) GAATCAGCCTCGCAACAAA (SEQ ID NO: 7) TTTGTTGCGAGGCTGATTC
  • Ad-shDicer genome prepared in 1) above was cleaved and washed with the restriction enzyme PacI present at the end of the viral genome, and transferred to HEK293 cells using the transfection reagent Lipofectamine2000 TM (Invitrogen). Transfected to produce Ad-shDicer.
  • Example 1-1 Confirmation of Ad Genome Proliferation Ability in Cancer Cells by Ad-shDicer
  • the amount of virus genome when Ad-shDicer prepared in Example 1 was allowed to act on various cancer cells was confirmed.
  • the cancer cells were confirmed for HeLa cells (human cervical cancer cells), SK HEP-1 cells (human hepatocellular carcinoma cells) and H1299 cells (human non-small cell lung cancer cells).
  • Ad-E3 (-) having the same genomic structure as Ad-shDicer prepared in Example 1 and expressing shRNA (shLuc) for firefly luciferase instead of shDicer Expressed Ad-shLuc was used.
  • Example 1-2 Confirmation of cell viability when Ad-shDicer was allowed to act The cell viability when Ad-shDicer prepared in Example 1 was allowed to act on various cancer cells and normal cells was confirmed. The control shown in Experimental Example 1-1 was confirmed in the same manner. In addition, a mock system without Ad was also used as a control. Cancer cells are HeLa cells (human cervical cancer cells), H1299 cells (human non-small cell lung cancer cells) and HepG2 cells (human hepatocellular carcinoma cells), normal cells are HUVEC (human umbilical vein endothelial cells), NHLF (human lung fibroblasts) and PrSC (prostate stromal cells) were confirmed.
  • HeLa cells human cervical cancer cells
  • H1299 cells human non-small cell lung cancer cells
  • HepG2 cells human hepatocellular carcinoma cells
  • normal cells are HUVEC (human umbilical vein endothelial cells), NHLF (human lung fibroblasts) and PrSC (prostate stromal cells) were confirmed.
  • Example 1-3 Confirmation of Dicer mRNA Level when Ad-shDicer was Acted
  • the cancer cells were confirmed for HeLa cells (human cervical cancer cells), H1299 cells (human non-small cell lung cancer cells) and SK HEP-1 cells (human hepatocellular carcinoma cells).
  • siRNA sequence for Dicer GAAUCAGCCUCGCAACAAA (SEQ ID NO: 1)
  • siRNA sequence for Dicer UUUGUUGCGAGGCUGAUUC (SEQ ID NO: 2)
  • Sequence of siRNA against Ago2 GCACGGAAGUCCAUCUGAA (SEQ ID NO: 3)
  • siRNA sequence for Ago2 UUCAGAUGGACUUCCGUGC (SEQ ID NO: 4)
  • siRNA sequence for XPO5 CUCGAUUGGAGAAGGUGUA (SEQ ID NO: 8) 6) siRNA sequence for XPO5: UACACCUUCUCCAAUCGAG (SEQ ID NO: 9)
  • SiRNA against Ago2 Dicer or XPO5 was introduced into HeLa cells (human cervical cancer cells) or H1299 cells (human non-small cell lung cancer cells).
  • HeLa cells human cervical cancer cells
  • H1299 cells human non-small cell lung cancer cells
  • control siRNA Allstars negative control siRNA; Qiagen
  • the Ad genome copy number was confirmed by quantitative PCR.
  • the copy number of the Ad genome was significantly increased compared to the control (see FIG. 7).
  • siRNA against XPO5 was allowed to act, the copy number of the Ad genome showed a value equal to or lower than that of the control (see FIG. 7).
  • SisiRNA against Dicer or Ago2 was introduced into each cell of HeLa cells or H1299 cells.
  • ControlAllsiRNA Allstars negative control siRNA
  • a VA-RNA expression plasmid was introduced.
  • the copy number of VA-RNA was measured by quantitative RT-PCR. That is, knocking down Dicer or Ago2 is thought to increase the copy number of full-length VA-RNA (see FIG. 9).
  • VA-RNA is transcribed in the cell nucleus, it is not a stage where it is transported out of the nucleus by XP05, or by suppressing the production of Ad-derived mivaRNA by introducing siRNA against Dicer or Ago2, etc. Alternatively, it was confirmed that by suppressing the activity of mivaRNA, an effect of promoting Ad proliferation can be obtained.
  • the shDicer expression cassette was introduced into H1299 cells (human non-small cell lung cancer cells), SK HEP-1 cells (human hepatocellular carcinoma cells) or HepG2 cells (human hepatocellular carcinoma cells).
  • H1299 cells human non-small cell lung cancer cells
  • SK HEP-1 cells human hepatocellular carcinoma cells
  • HepG2 cells human hepatocellular carcinoma cells
  • the Ad genome copy number was confirmed by quantitative PCR.
  • the copy number of the Ad genome was significantly increased compared to the control (see FIG. 10). From this, it was confirmed that by suppressing the production of Ad-derived mivaRNA with shDicer, the effect of promoting Ad growth can be obtained.
  • Ago2 or Dicer expression plasmids were introduced into HeLa cells (human cervical cancer cells) or H1299 cells (human non-small cell lung cancer cells).
  • the control was a cell into which a control plasmid (p3 ⁇ FLAG-CMV10) that expresses nothing was introduced.
  • the wild-type Ad solution was infected at 37 ° C. for 24 hours so that the MOI was 5%.
  • the Ad genome copy number was confirmed by quantitative PCR.
  • the copy number of the Ad genome was significantly reduced compared to the control (see FIG. 11).
  • Example 2 Cell killing effect of Ad-shDicer on each cancer cell
  • Ad-shDicer prepared by the same method as in Example 1 was used for each of HeLa cells (human cervical cells) or H1299 cells (human non-small cell lung cancer cells). The cell viability when acting on cancer cells was confirmed.
  • the control shown in Experimental Example 1-1 was confirmed in the same manner.
  • various virus suspensions prepared using the culture medium are allowed to act at MOI 0.01, 0.1, 1, 10, 100, and 37 ° C. For 24 hours. Thereafter, the virus solution was removed, a culture solution was added, and the mixture was cultured at 37 ° C.
  • the Ad-shDicer of the present invention showed a higher cell killing effect than the control Ad-E3 ( ⁇ ) and Ad-shLuc (see FIG. 12).
  • Example 3 Anti-cancer effect of Ad-shDicer on each cancer cell (anti-tumor effect)
  • the control shown in Experimental Example 1-1 was confirmed in the same manner.
  • Ad-shDicer of the present invention has a higher antitumor activity than the control Ad-E3 ( ⁇ ) and Ad-shLuc (see FIG. 13).
  • VA-RNAI and II expression plasmids (pAdvantage) -DNaeI, pAdVantage, VA-RNAI expression plasmid (pVAI) or VA-RNAII expression plasmid (pVAII) are transfected, and VA-RNA (I and II), VA-RNAI or HeLa cells overexpressing VA-RNAII were cultured for 48 hours, and the expression levels of miR-27a and miR-27b were measured by quantitative RT-PCR. As a result, it was shown that the expression levels of miR-27a and miR-27b decreased due to overexpression of VA-RNA, respectively (see FIG. 15A).
  • HeLa cells were transfected with a VA-RNA expression plasmid and a reporter plasmid (psiCHECK-2-mR-27aT or -miR-27bT) containing 2 copies of the miR-27a or miR-27b complementary sequence in the 3'-UTR of the RLuc gene And cultured for 48 hours. Thereafter, luciferase activity was measured. As a result, it was shown that the expression levels of miR-27a and miR-27b were decreased by VA-RNA (see FIG. 15B).
  • Example 4 Preparation of Ad loaded with a tough decoy RNA expression cassette that inhibits miR-27a, b
  • a tough decoy RNA against miR-27a or miR-27b (hereinafter referred to as "TuD- 27a or TuD-27b "), and the production of an oncolytic Ad carrying a cassette capable of expressing siRNA against miR-27a or miR-27b will be described.
  • the Ad in this example is referred to as “Oncolytic Ad-TuD-27a” or “Oncolytic Ad-TuD-27b”.
  • the genomic structure of the produced Oncolytic Ad-TuD-27a or Oncolytic Ad-TuD-27b is as shown in FIG.
  • E1 It was constructed based on a vector plasmid that lacks the region (342-3523) and the E3 region (28133-30818) and can insert a foreign gene into the E1-deficient region.
  • the hTERT promoter which is a tumor-specific promoter
  • E1A, IRES, E1B, and polyA (pA) signal sequences were ligated and mounted on the E1 deficient region of the vector plasmid pAdHM19 to obtain an Oncolytic Ad genome.
  • TuD-27a or TuD-27b an expression cassette for TuD-27a or TuD-27b was mounted in the E3-deficient region.
  • the sequence corresponding to TuD-27a or TuD-27b used in this example consists of the base sequence shown in SEQ ID NO: 12 or 13.
  • the underlined portion is an antisense sequence against miR-27a or miR-27b.
  • TuD-27a GACGGCGCUAGGAUCAUCAAC GCGGAACUUAGAUCUCCACUGUGAA CAAGUAUUCUGGUCACAGAAUACAAC GCGGAACUUAGAUCUCCACUGUGAA CAAGAUGAUCCUAGCGCCGUCUU (SEQ ID NO: 12)
  • TuD-27b GACGGCGCUAGGAUCAUCAAC GCAGAACUUAGAUCUCCACUGUGAA CAAGUAUUCUGGUCACAGAAUACAAC GCAGAACUUAGAUCUCCACUGUGAA CAAGAUGAUCCUAGCGCCGUCUU (SEQ ID NO: 13)
  • Oncolytic Ad-TuD-27a or Oncolytic Ad-TuD-27b The TuD-27a or TuD-27b genome produced in 1) above is cleaved with the restriction enzyme PacI present at the end of the viral genome, washed, and transferred.
  • Oncolytic Ad-TuD-27a or Oncolytic Ad-TuD-27b was prepared by transfecting HEK293 cells using the injection reagent Lipofectamine2000 TM (Invitrogen).
  • Example 4-1 Confirmation of cell viability when Oncolytic Ad-TuD-27a or Oncolytic Ad-TuD-27b was allowed to act Oncolytic Ad-TuD-27a or Oncolytic Ad-TuD-27b produced in Example 4 The cell viability was confirmed when this was allowed to act on HeLa cells or H1299 cells.
  • the control (conventional Oncolytic Ad) shown in Ad-E3 (-) that does not express any tough decoy RNA was also confirmed in the same manner.
  • a mock system without Ad was also used as a control.
  • Example 4-2 Confirmation of Ad Genomic Proliferation Ability in Cancer Cells by Oncolytic Ad-TuD-27a or Oncolytic Ad-TuD-27b Oncolytic Ad-TuD-27a or Oncolytic Ad-TuD- produced in Example 4 The amount of viral genome when 27b was allowed to act on HeLa cells or H1299 cells was confirmed. The control (conventional Oncolytic Ad) was confirmed in the same manner as in Experimental Example 4-1.
  • the Ad of the present invention can promote the proliferation of Ad more effectively than the conventional Ad.
  • the antitumor agent containing Ad of the present invention as an active ingredient proliferation is effectively promoted in the target tumor cells, and an antitumor effect is exhibited. Thereby, a more effective antitumor agent can be provided and useful.
  • Ad contained as an active ingredient in the antitumor agent of the present invention has a growth promoting ability in a desired target cell, and therefore can be used at a lower concentration than normal restricted growth Ad, and is also economically useful. .
  • the proliferation promoting method of the present invention it is possible to effectively promote the proliferation of Ad as compared with the conventional method. If Ad can be effectively propagated in a desired cell such as a target tumor cell, an antimalignant effect such as tumor lysis can be effectively exerted. Then, by applying the growth promotion method of the present invention to an Ad production system in cells, Ad can be produced more effectively, which is useful.

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Abstract

Le but de la présente invention concerne un adénovirus (Ad) qui peut être cultivé efficacement à l'aide de cellules souhaitées telles que des cellules tumorales cibles. L'invention concerne un procédé pour favoriser la croissance de l'Ad par lequel l'Ad est cultivé efficacement à l'aide des cellules souhaitées. L'invention concerne en outre des cellules d'encapsidation pour la production de l'Ad pouvant cultiver l'Ad efficacement. L'Ad pouvant être cultivé efficacement à l'aide de cellules souhaitées telles que des cellules tumorales cibles est produit par l'inhibition de la fonction de microARN VA dérivé de l'Ad dans le génome de l'Ad ou par le blocage de miR-27a et/ou miR-27b dans le génome de l'Ad. L'inhibition de la fonction de microARN VA dérivé d'Ad ou le blocage de miR-27a et/ou miR-27b est obtenue par le blocage par Dicer et/ou Ago2.
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WO2020166727A1 (fr) * 2019-02-12 2020-08-20 国立大学法人大阪大学 Virus oncolytique utilisant l'adénovirus humain de type 35 comme base
JPWO2020166727A1 (ja) * 2019-02-12 2021-12-16 国立大学法人大阪大学 ヒト３５型アデノウイルスを基盤とした腫瘍溶解性ウイルス
JP7508109B2 (ja) 2019-02-12 2024-07-01 国立大学法人大阪大学 ヒト３５型アデノウイルスを基盤とした腫瘍溶解性ウイルス
JP2022535039A (ja) * 2019-05-30 2022-08-04 伍沢堂 組換え腫瘍溶解性ウイルスとその調製方法、使用および医薬品
JP7441245B2 (ja) 2019-05-30 2024-02-29 伍沢堂 組換え腫瘍溶解性ウイルスとその調製方法、使用および医薬品

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