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WO2020124525A1 - Utilisation de mir-7 pour la préparation d'un médicament anti-rotavirus - Google Patents

Utilisation de mir-7 pour la préparation d'un médicament anti-rotavirus Download PDF

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Publication number
WO2020124525A1
WO2020124525A1 PCT/CN2018/122565 CN2018122565W WO2020124525A1 WO 2020124525 A1 WO2020124525 A1 WO 2020124525A1 CN 2018122565 W CN2018122565 W CN 2018122565W WO 2020124525 A1 WO2020124525 A1 WO 2020124525A1
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mir
rotavirus
nsp5
virus
preparation
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Chinese (zh)
Inventor
周艳
李鸿钧
陈林林
解裕萍
胡晓青
吴晋元
尹娜
孙茂盛
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Institute of Medical Biology of CAMS and PUMC
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Institute of Medical Biology of CAMS and PUMC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the application of the present invention relates to the field of biomedicine, especially the application of MicroRNA-7 in the preparation of anti-rotavirus drugs.
  • Rotavirus is one of the main pathogens that cause diarrhea in infants and young children under five years old.
  • the number of gastroenteritis cases in children caused by RV infection in the world still reaches about 125 million each year, resulting in the death of about 400,000 children.
  • miRNAs and siRNAs can play an important role in many biological processes because they can regulate the post-transcriptional expression of genes.
  • the miRNA can bind to the complementary sequence of the target mRNA, thereby triggering the degradation or translation inhibition of the target mRNA molecule. It has found a new way to inhibit the expression of disease-causing genes, and has great potential for the development of small RNA drugs.
  • MicroRNAs are a class of non-coding small RNAs of about 22 bases in length. They control the expression of more than one-third of genes in eukaryotes at the post-transcription level by inhibiting translation and causing mRNA degradation. Almost all biological processes such as cell differentiation and development, proliferation, apoptosis and metabolism. In the process of virus infection, cells can suppress the infection and proliferation of foreign invading viruses by modulating the host cell's microRNA to target viral genes or their own signaling pathway genes. MicroRNA-7 (miR-7) is a small RNA that was discovered in 2001 and has evolved conservatively.
  • miR-7 In human cells, the primary transcription precursor of miR-7 (pri-miR-7) is encoded by chromosomes 9, 15 and 19, transported and finally processed into the same mature 23-base miR -7 sequence.
  • Zhang et al. found that siRNAs synthesized in vitro specifically targeting poliovirus (PV) 5'-UTR can not only effectively inhibit the replication of PV and virus titers, but also significantly promote miR-7 in host cells. The expression is up-regulated.
  • PV poliovirus
  • miR-7 can affect the replication of white spot syndrome virus (WSSV) in crabs by targeting and suppressing the expression of immune-related factor Myd88.
  • WSSV white spot syndrome virus
  • the interaction between the virus and the host cell is a regulation strategy of the virus and the host cell formed during the long-term evolution.
  • the cell can invade the virus by regulating the microRNA of the host cell to target the virus gene or its own signaling pathway gene.
  • the infection and proliferation are suppressed.
  • the purpose of the present invention is to provide the application of MicroRNA-7 in the preparation of anti-rotavirus drugs.
  • the present invention finds that miR-7 has a significant regulation target gene NSP5 (SED NO.3), miR-7 targets 17bp-36bp of the NSP5 gene of the rotavirus ZTR-68 strain, and miR-7-NSP5 functions
  • NSP5 significant regulation target gene
  • miR-7-NSP5 functions The locus can be used as a new and effective anti-rotavirus therapeutic target.
  • miR-7 small RNA mimics in the preparation of anti-rotavirus drugs.
  • the miR-7 small RNA mimic is mml-miR-7 sense single-stranded oligonucleotide.
  • the sense single-stranded oligonucleotide of miR-7 can be artificially synthesized or directly commercially available.
  • the sense single-stranded oligonucleotide of miR-7 can also be modified by PNA, LNA or 2-Ome.
  • the registration number of miR-7 is (MI0007581, http://www.miRbase.org).
  • the precursor of miR-7 is miR-7-1, and the accession number is (MIMAT0006159, http://www.miRbase.org).
  • the isolated wild-type rotavirus ZTR-68 strain was infected with sensitive cells MA104 cells for deep sequencing analysis, and the microRNAs expression profile during rotavirus infection was analyzed, and it was found that miR-7 was infected. After the virus was increased by nearly 400 times. Through qRT-PCR verification, we found that miR-7 was indeed upregulated nearly 20-fold after rotavirus ZTR-68 infection. After different types of rotavirus strains infected MA104 cells, miR-7 expression was up-regulated to varying degrees. ZTR-68 strain rotavirus and its expression level gradually increased with the time of viral infection and the amount of MOI infected.
  • MiR-7 can combine with the NSP5 gene to affect the expression of NSP5 protein, affect the formation of the virus pool during virus replication, and further inhibit virus replication. .
  • miR-7 can indeed significantly affect the replication of the virus and suppress the diarrhea caused by the viral infection after being introduced into the intestinal tract of suckling rats.
  • Rotavirus belongs to the genus Reovirus, and is an encapsulated icosahedral virus.
  • the genome is discontinuous and consists of 11 segments of double-stranded RNA, encoding 6 structural proteins (VP1-4, 6, 7) and 6 non-structural proteins (NSP1-6).
  • Rotavirus non-structural protein NSP5 is encoded by the 11th gene in the viral RNA genome and is a highly phosphorylated dimeric protein rich in threonine (25% of total amino acids) and serine residues.
  • the molecular weight after translation modification conditions is 26-35Kd.
  • NSP5 rich in serine-threonine shifts between hypophosphorylated hyperphosphorylated isomers during the replication cycle and participates in the virus pool
  • the virus pool is a place for rotaviruses to prevent the innate immune monitoring of genome replication, which is very important for virus replication.
  • the microRNA that inhibits rotavirus replication with NSP5 as the target has not been discovered and reported.
  • rotavirus NSP5 is the target of miR-7, and the combination of miR-7 and NSP5 can inhibit rotavirus replication.
  • Over-expression mimics of miR-7 introduced into suckling mice can inhibit rotavirus replication and diarrhea caused by rotavirus infection.
  • RNA interference technology has been widely used in various antiviral studies, such as HIV-1, HCV and influenza viruses.
  • miR-7 has the potential to become an anti-rotavirus replication small RNA drug, and the miR-7-NSP5 action site may be a new and effective anti-rotavirus therapeutic target.
  • FIG. 1 Time-sensitive expression detection of miR-7: After ZTR-68 infected MA104 cells at different time periods (0, 12h, 24h, 36h and 48h), RT-PCR was used to detect the expression level of miR-7 (*p ⁇ 0.01)
  • Figure 2 RT-PCR detection of miR-7 expression level after infection of MA104 cells with different MOI (0.1, 0.5, 1 and 2) ZTR-68 rotavirus
  • Figure 3 RT-PCR detection of miR-7 expression level after infection of MA104 cells infected with different G-type rotavirus with 0.5MOI
  • Figure 4 RT-PCR detection of miR-7 expression level after 36 hours of ZTR-68 infection in HT-29 cells and Caco-2 cells
  • FIG. 6 Immunofluorescence to detect the effect of miR-7 up-regulation on rotavirus replication
  • A Rotavirus-specific immunofluorescence to detect the effect of miR-7 up-regulation on viral protein expression (red is RV, blue is DAPI );
  • B Statistics of positive cells in rotavirus immunofluorescence test results after up-regulation and down-regulation of miR-7 molecules (average of 10 random fields)
  • Figure 8 Analysis of the relationship between miR-7 and rotavirus NSP5 gene target
  • A miRanda software predicts the binding of miR-7 to rotavirus NSP5 gene
  • B Dual luciferase reporter system detects miR-7 and NSP5 Construction of mutant plasmids
  • C Dual luciferase reporter system to detect the binding of miR-7 to NSP5 (**P ⁇ 0.05)
  • FIG. 10 Westernblot detection of NSP5 eukaryotic expression plasmid and miR-7 mimic co-transfected 293 cells to detect the expression of NSP5 protein
  • Figure 13 HE staining to observe the small intestinal villi lesions in neonatal rats after miR-7 up-down regulation
  • Example 4 The role of miR-7 in rotavirus replication
  • Example 5 miR-7 targeting rotavirus NSP5 gene regulates virus proliferation
  • the dual luciferase reporter system was used to detect the regulatory effect between miR-7 and the target gene NSP5: we cloned the target sequence (NC) and mutant sequence (NSP5Mut) of the target gene NSP5 into the luciferase expression plasmid, and miR -7 molecules of mimics and control microRNA sequences were co-transfected into HEK293 cells, and the relative change of luciferase activity was detected by luciferase detection kit to identify whether miR-7 had a regulatory effect on the target gene. The results showed that miR-7 It can significantly regulate the expression of luciferase with the target gene NSP5. After the binding site was mutated, the regulatory relationship disappeared, indicating that miR-7 regulates the expression of NSP5 through this binding site ( Figure 8B, 8C).
  • miR-7 can regulate the expression of NSP5 gene.
  • pEGFP-N2-NSP5 eukaryotic expression vector When co-transforming MA104 cells with miR-7 mimics and inhibitor for 48h, we can find that miR-7 -7 can significantly inhibit the expression of rotavirus NSP5 protein (Figure 10).
  • NSP5 is the main regulatory protein that forms the rotavirus virus pool
  • the NC group after up-regulating miR-7, the number of intracellular virus pools was reduced, and the structure of the virus pool was incomplete. The number of intracellular virus down-regulating miR-7 was higher, and the number and morphology of virus pools were not significantly different from NC ( Figure 11) ).
  • Example 6 The role of miR-7 in rotavirus infection model
  • the miR-7 overexpression mim-7 mimir-7 and miR-7 expression inhibitor miR-7 are purchased from Shanghai Gemma Gene Company, miR-7 mimir-7 and expression inhibitor miR-7 antagomir was purchased from Guangzhou Ruibo Biotechnology Co., Ltd.
  • miR-7 agonist miR-7 a miRNA agonist for animal experiments with special chemical modification
  • miR-7 antagomir a miRNA antagonist for animal experiment with special chemical modification
  • miRNAnegativecontrol with special chemical modification Administered by gavage. 24 hours after dosing, 200 ID50 of rotavirus was intragastrically infected, and normal feeding was observed. According to the scoring rules of BOSHUIZEN JA and others for diarrhea in suckling rats, the diarrhea in suckling rats is scored (0 to 4) according to the color, hardness, quantity, etc.
  • the score of no fecal discharge is 0, and the score of brown shaped stool Points, brown soft stool score 2 points, yellow soft stool score 3 points, yellow watery stool score 4 points, perianal fecal pollution score 4 points, greater than 2 points are considered to have diarrhea.
  • the diarrhea score and diarrhea rate of each group of suckling rats were counted.
  • the NC group and the miR-7 antagomir group had significant diarrhea after 24 hours of challenge, with a score of 4 points, and the miR-7 antagomir group had a higher degree of diarrhea than the NC group. There was no significant diarrhea in the miR-7 agomir group after challenge.
  • the suckling rats were euthanized, and their intestinal tissues were taken for HE staining to detect pathological changes.
  • the intestinal villi showed obvious swelling and damage, and a large number of vacuolated cells were visible on the top of the intestinal villi.
  • the small intestinal villi in the antagomir group that down-regulated miR-7 also showed obvious swelling and breakage, and a large number of vacuolated cells were visible on the top of the small intestine villi, which was more severe than the NC group.
  • miR-7 In the miR-7's agomir group, the small intestinal villi showed slight swelling and inflammatory cell infiltration, but no damage was found (Figure 13). Combined with diarrhea scores, miR-7 can significantly inhibit rotavirus replication and reduce or suppress diarrhea caused by rotavirus infection in suckling mice.

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Abstract

Utilisation de miR-7 pour la préparation d'un médicament anti-rotavirus. Il a été découvert que le rotavirus NSP5 est une cible de miR-7, et la combinaison de miR-7 et NSP5 peut inhiber la réplication du rotavirus. L'introduction d'analogues de miR-7 sur-exprimés dans l'organisme d'une souris allaitante peut inhiber la réplication du rotavirus et empêcher la diarrhée provoquée par une infection à rotavirus, c'est pourquoi le miR-7 peut être utilisé pour la préparation d'un médicament anti-rotavirus.
PCT/CN2018/122565 2018-12-21 2018-12-21 Utilisation de mir-7 pour la préparation d'un médicament anti-rotavirus Ceased WO2020124525A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009026576A1 (fr) * 2007-08-23 2009-02-26 Keren Pharmaceuticals Ciblage d'arn avec séquences de guide externes
CN104342439A (zh) * 2013-07-23 2015-02-11 中国科学院遗传与发育生物学研究所 miR-7及其应用
WO2017081110A1 (fr) * 2015-11-09 2017-05-18 Curevac Ag Vaccins contre les rotavirus
CN108728530A (zh) * 2018-06-21 2018-11-02 浙江大学 一种鉴定缺血性卒中病因亚型的microRNA标志物组合及其用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009026576A1 (fr) * 2007-08-23 2009-02-26 Keren Pharmaceuticals Ciblage d'arn avec séquences de guide externes
CN104342439A (zh) * 2013-07-23 2015-02-11 中国科学院遗传与发育生物学研究所 miR-7及其应用
WO2017081110A1 (fr) * 2015-11-09 2017-05-18 Curevac Ag Vaccins contre les rotavirus
CN108728530A (zh) * 2018-06-21 2018-11-02 浙江大学 一种鉴定缺血性卒中病因亚型的microRNA标志物组合及其用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SHI, J.: "Identification and validation of a novel microRNA-like molecule derived from a cytoplasmic RNA virus antigenome by bioinformatics and expe- rimental approaches", VIROLOGY JOURNAL, 1 July 2014 (2014-07-01), XP021190862 *
ZHOU, Y.: "MicroRNA Profile Analysis of Host Cells Before and After Wild Human Rotavirus Infection", JOURNAL OF MEDICAL VIROLOGY, 11 March 2016 (2016-03-11), XP055721640 *

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