WO2004016780A1 - Vecteur de virus adeno-associe inhibant le fonctionnement du gene par le rnai - Google Patents

Vecteur de virus adeno-associe inhibant le fonctionnement du gene par le rnai Download PDF

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WO2004016780A1
WO2004016780A1 PCT/CN2003/000685 CN0300685W WO2004016780A1 WO 2004016780 A1 WO2004016780 A1 WO 2004016780A1 CN 0300685 W CN0300685 W CN 0300685W WO 2004016780 A1 WO2004016780 A1 WO 2004016780A1
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rnai
recombinant
psnav
lucrnai
plasmid
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PCT/CN2003/000685
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Xiaobing Wu
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Xiaobing Wu
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/14—Type of nucleic acid interfering N.A.
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/12—Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention belongs to the field of biotechnology invention. Specifically, it involves carrying a RNAi nucleotide fragment with a plasmid, and transfecting the plasmid carrying RNAi into an in vitro cultured cell or animal, so that the RNAi nucleotide fragment can be continuously replicated in the cell or animal to achieve The biological role of RNAi nucleotide fragments.
  • the RNAi nucleotide fragment is also packaged into a recombinant adeno-associated virus vector carrying the RNAi nucleotide fragment, and the gene transfer and replication of the RNAi nucleotide fragment are performed through the adeno-associated virus vector to achieve the RNAi nucleoside.
  • Gene silencing (GENE SILENCING) phenomenon exists widely in plants, fungi and animals. Due to the rise of transgenic technology, people have conducted more in-depth research on transgene silencing. About 30% of transgenic experiments in higher plants have resulted in gene silencing, which is a universal gene regulation mechanism.
  • transgene silencing There are two types of transgene silencing: one is transcriptional gene silencing (TGS), and the other is posttranscriptional gene silencing (PTGS).
  • TGS transcriptional gene silencing
  • PTGS posttranscriptional gene silencing
  • the former A C was born in the nucleus, while the latter occurred in the cytoplasm. Both are related to hypermethylated.
  • TGS phenomenon methylation mainly occurs in the promoter region, and gene transcription is suppressed.
  • PTGS methylation mainly occurs in the coding region of genes.
  • Post-transcriptional Shen A ⁇ is now a sequence-specific RNA degradation process, which mainly acts on transcription products with higher homology, including transcription products of endogenous genes with homology. It is widely found in various organisms. Until recently, it has not been found that it is called cosuppression in plants, Banling in fungi, and RNA interference in animals. Phenomenon may be very similar, both are related to PTGS Related phenomena. PTGS is now generally considered to be a defense system against viruses, transposable elements and other transferable nucleic acids formed by organisms during long-term evolution. The introduction of these exogenous nucleic acids may fatally disrupt the homeostasis mechanism in the host cell.
  • transgene silencing is nothing more than the host cell's view of the foreign gene as a sequence harmful to itself and suppressing it.
  • This inhibition of cells against foreign nucleic acids has a sequence-specific characteristic, so once the intra-transcriptional silencing mechanism is activated, the cells have "immunity" to the homologous sequences of the transferred nucleic acids.
  • RNAi first observed in caenorhabditis nematodes and fruit flies
  • dsRNA double-stranded RNA
  • ssRNA target single strand RNA
  • the enzyme responsible for cleaving dsRNA has been proven to be a dsRNA-specific RNase, called Dicer, which produces dsRNA degradation products.
  • dsRNAs or short interfering R As serve as guides for the enzyme complexes required to degrade the target ssRNA, and it includes 21-23 wicks in the regular gaps in the region corresponding to the input dsRNA.
  • Dicer also has helicase activity, and functions in other regions have not yet been determined. These are important for RNAi in Drosophila and nematodes. Moreover, Dicer plays a role in the processing of transcripts required for normal development. So far, only Argonaute2, a protein component in multi-subunit complexes or RA-mediated cleavage target RNA silencing complexes (RISC), has been confirmed.
  • dsRNA triggers gene silencing in mammalian somatic cells. Small synthetic dsRNA or siR A can avoid this non-specific suppression of gene expression in mammalian cells. Study RNAi as a way to control gene expression and explore gene function at the genome level.
  • RNA interference is an evolutionarily conserved genome-level immune monitoring mechanism. It is a multi-step process and involves the generation of active small 21-23nt interference through the action of the RNase HI endonuclease Dicer. of RNA (siRNA), which mediates complement-specific degradation of homologous mRNA sequence 1, 2.
  • siRNA RNA
  • the RNAi phenomenon is widely found in most eukaryotes such as fungi, Arabidopsis, leech, planaria, trypanosomes, zebrafish and so on. The mechanism of RNAi is being elucidated gradually. At the same time, as a powerful tool in the field of functional genomics, RNAi is becoming more and more important. The most interesting things about RNAi are:
  • dsRNA is double-stranded RNA, not single-stranded antisense R A. It is an interference reagent.
  • Intervention activity can cause interference in cells and tissues far from the site of introduction.
  • dsRNAs The response of mammalian cells to dsRNAs can activate two pathways: extensive non-sequence-specific mRNA degradation and translation shutdown caused by dsRNAs over 30bp, and short dsRNA molecules 19-23bp (short dsRNA molecules, siRNA) synthesized in vitro Mimicking RNAi pathway intermediates, can cause sequence-specific gene expression inhibition in mammals 3
  • Viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, and the like.
  • adenovirus-associated virus deno-associated virus (AAV) vector has attracted attention due to its safety, stability, infection of both dividing and non-dividing cells, high infection efficiency, and long-term expression of foreign genes.
  • One or more copies of the recombinant AAV vector DNA are stably integrated in the genome of the recombinant AAV vector cell, which contains the two ITR sequences of the AAV virus and the therapeutic gene expression unit located therebetween.
  • Therapeutic gene expression unit consists of eukaryotic expression promoters (such as HCMV IE promoter, SV40 promoter, globin gene promoter, inducible promoter, various tissue-specific promoters, etc.), therapeutic gene, mRNA tailing signal , The length does not exceed 5.0kb.
  • HSVl-rc virus entered the cell and its DNA was replicated in large quantities and eventually produced. Progeny virus.
  • Carrying rep / cap genes are also synchronized copy HSVl-rc viral DNA replication, resulting in re p / ca p high copy gene.
  • the rep gene encodes 4 types of Rep proteins (Rep78, Rep68, Rep52, Rep40), which enables the recombinant AAV vector DNA to be rescued from the cell genome and replicated in large quantities to eventually be single-stranded and packaged into AAV capsids;
  • the cap gene encodes 3 types of shells
  • the proteins VP1, VP2, and VP3 assemble into capsids in the nucleus. Summary of the invention
  • a plasmid vector expressing short hairpin RA (shR A), which is intended to carry a variety of RNAi;
  • a plasmid vector is designed and constructed to carry luciferase shRNA
  • the expression of this luciferase shRNA is controlled by the human U6 snRNA promoter (a DNA-directed RNA Pol III extragene promoter).
  • the advantage of Pol III is to guide the synthesis of small non-coding transcripts whose 3 'terminus terminates At a string of 4-5 T's.
  • the invention relates to the use of a plasmid to carry RNAi nucleotide fragments, and the plasmid carrying RNAi is transfected into cells or animals cultured in vitro, so that the RNAi nucleotide fragments can be continuously transcribed in cells or animals.
  • the transcription unit composed of the RNAi nucleotide fragment and its upstream and downstream regulatory sequences is constructed by constructing an AAV vector cell line and a dye capable of packaging and producing a recombinant virus (rAAV-LucRNAi) carrying the RNAi nucleotide transcription unit.
  • Virus and other processes are also packaged into a recombinant viral vector carrying RNAi nucleotide fragment transcription units, and gene transfer and transcription of RNAi nucleotide fragments are performed through adeno-associated virus vectors to achieve RNAi nucleotide fragments The biological role that should be played.
  • a short hairpin loop expression plasmid in the examples of the present invention, we constructed a short hairpin loop expression plasmid in
  • BHK-21 cells inhibited luciferase expression.
  • luciferase RNAi expression plasmid pSNAV / U6 / LucRNAi in the description of the present invention pSNAV / U6 / LucRNAi is the same as pSNAV-U6-LucRNAi.
  • the aforementioned AAV vector plasmid pSNAV can also be replaced by a series of general-purpose AAV vectors (Chinese patent application: 99119038.6). BHK-21 cells were co-transfected with pMAMneoLuc plasmid, and luciferase cell lines were transfected separately, and their inhibitory effects on luciferase expression were measured.
  • Figure 1 shows the PCR amplification results of human U6 snRNA promoter, where M is DL2000 Marker; 1 is the PCR product of human U6 snRNA promoter.
  • Figure 2 shows the results of electrophoresis of Xhol + Xmnl digestion and identification of recombinant plasmid pSNAV-U6-LucRNAi, where M is the molecular weight marker of DL2000 + DL15000; 1 is the result of Xhol + Xmnl digestion of pSNAV / U6 / Luc; 2 is of pSNAV Xhol + Xmnl digestion control.
  • FIG. 3 Construction of rAAV vector plasmid pSNAV-U6-LucR Ai containing a short hairpin loop RNA expression cassette.
  • Figure 4 pSNAV-U6-LucRNAi inhibits the level change of co-transfected plasmid pMAMneoLuc luciferase.
  • Plasmids, forests and cell lines The plasmid pMAMneoLUC was purchased from InVitrogen.
  • the universal AAV vector plasmid pSNAV was constructed by Wu Xiaobing et al. The above plasmids were all in E. coli DH5 strain
  • T4 ligase and various restriction enzymes are products of Biolab, and liposome transfection agent Lipofectamine was purchased from GIBCO / BRL.
  • T4 ligase and various restriction enzymes are products of Biolab, and liposome transfection agent Lipofectamine was purchased from GIBCO / BRL.
  • Obtaining of human U6 snRNA promoter ⁇ Extracting human genomic DNA from peripheral blood. Vector NTI was used to design primers.
  • the upstream primer sequence (labeled as P1) was: CTCGAGCCCCAGTGGAAAGACGCG, and the downstream primer sequence (labeled as P2) was: CGTGTCATCCTTGCGCAGGGG (: The length of the amplified product was: 414bp.
  • the upstream primer and the internal primer were used to perform nested PCR to amplify the 334bp (-334bp- + lbp) human U6 promoter sequence.
  • the internal primer sequence (labeled as P3) was: AAGCTTGAAGTGTTTCGTGGTTTCCACA.
  • the nested PCR product was loaded into T of Promeg Corporation.
  • Xhol and Hind III were used to cut the 330bp human U6 promoter sequence.
  • the plasmid was inserted into the pShuttle / Xhol + Hindlll digested plasmid to construct the pShuttle-U6 plasmid.
  • the PCR amplification results of the human U6 snR A promoter sequence (-334nt — lnt) are shown in FIG. 1 in the accompanying drawings, and its nucleotide sequence is shown in Table 1.
  • A is the human U6 snRNA promoter sequence published in PubMed;
  • B is the human U6 snRNA promoter sequence we isolated, labeled P6.
  • Table 1 Our isolated human U6 snRNA promoter sequences are aligned with those published in PubMed
  • the PCR reaction system is as follows: template 1 ⁇ , Taq 0.5 ⁇ I, 10X PCR buffer 1 ⁇ I, U6 upstream primer 1 ⁇ , U6 internal primer 1 ⁇ 1, dH 2 0 41.5 ⁇ K 94 ° C 5 minutes, 94 ° C 45 seconds, 56 "C 45 seconds, 72 ° C 45 seconds, 30 cycles.
  • the recombinant plasmids with correct digestion results were expanded with 330bp promoter.
  • the construction of the AAV vector plasmid pSNAV-U6-lucRNAi is shown in the instruction manual. Figure 3 of the figure. Example 3 Observation of the inhibitory effect of pSNAV-U6-LucRNAi transfection on co-transfected pMAMneoLuc expression of luciferase
  • A is blank: the result of the white control group
  • B is the result of the pSNAV / U6 / Luc group
  • C is the result of the pMAMneoLuc group
  • D is the result of the pMAMneoLuc + pSNAV / U6 / Luc group.
  • the expression of luciferase was detected at 12, 24, and 48 hours after co-transfection.
  • the inhibition effect was enhanced from 12 to 24 hours, the inhibition effect was highest at 24 hours, and the inhibition effect was weakened after 48 hours. (Results not shown). It shows that with time, as the transfected inhibitory plasmid, that is, the luciferase shRNA expression plasmid pSNAV-U6-LucRNAi, is diluted with cell division, the inhibitory effect decreases with time.
  • the luciferase expression detected at 24 hours after transfection is shown in Table 2.
  • Detection kit Luciferase Assay Syatem (Promega, Cat. # E1501), detection instrument Turner Design Instument 9600-001 (Turner BioSystems, USA). Detection steps: 2.1 Cell luciferase protein extraction: Wash the test cells twice with PBS without Mg 2+ Ca 2+ , and remove PBS as much as possible, add lxLysis Buffer (cell lysate) 100 ⁇ 1 ( (24-well plate), after the cells completely detached, suck them into a 1.5ml Eppendorf tube, vortex for 15 seconds, centrifuge at 4 ° C at 12000 rpm for 4 minutes, and take the supernatant for immediate detection or freezing at -70 ° C for later use.
  • lxLysis Buffer cell lysate
  • the prepared luciferase reaction substrate is first equilibrated to about 25 ° C at room temperature. Add 96 ⁇ l, 100 ⁇ l of luciferase reaction substrate to each well of the 96-well plate for detection. After vortexing slightly, use a single reading. The detection value is Relative Light Units (RLU) 0
  • pMAMneoLuc was transfected into BHK-21 cells with liposome Lipofectamine 2000. After 48 hours, selective culture was performed with 800 ⁇ g / ml G418. After the resistant clones are formed (about 10 days), trypsinize and mix them. Continue to grow with G418 for 15 days. After passage, switch to culture medium without G418. Name this cell forest BHK. / Luc cells.
  • A is the result of the blank control group
  • B is the result of the 0. ⁇ ⁇ group
  • C is the result of the 0.5 ⁇ 1 group
  • D is the result of the 1 ⁇ ⁇ group.
  • the results of inhibition of luciferase expression in luciferase cell lines by pSNAV-U6-LucRNAi are shown in Table 3.
  • the 3 ⁇ ⁇ inhibitory plasmid reduced the expression of luciferase to 70% as much as possible. This shows that shRNA-mediated RNAi synthesized from DNA templates in vivo has a dose effect. The more RNAi plasmids are transfected, the stronger the inhibitory effect.
  • Example 4 Establishment of AAV vector cell line containing LucR Ai gene and capable of packaging and producing recombinant virus rAAV-LucRNAi
  • pSNAV-U6-LucRNAi was transfected into BHK-21 cells with liposome Lipofectamine 2000. After 48 hours, the cells were selectively cultured with 800 ⁇ g / ml G418. After the resistant clones are formed (about 10 days), trypsinize and mix them, continue to use G418 to select and grow until the cells are full. After passage, use G418-free medium to culture. This cell line is named BHK / LucRNAi. .
  • the AAV vector Lin cells BHK / LucRNAi expansion culture the culture broth containing 10% fetal bovine serum RPMI1640.
  • a spinner flask 110mmX480mm, a product of Wheaton
  • the cells are full (about 8X10 8 cells per bottle), discard the culture solution.
  • MOI helper virus
  • the rAAV-LucRNAi virus titer was determined with reference to 9 '10 and was performed by dot hybridization.
  • Example 7 In vitro inhibition experiment of rAAV-LucRNAi virus
  • each cell was seeded with 2.5 ⁇ 10 4 BHK-21 / LucRNAi cells, and the cells were infected with rAAV-LucRNAi virus (lXlo g./well) 24 hours later, and then changed to 10% RPMI 1640. The luciferase activity was measured at 27 hours.
  • rAAV-LucRNAi virus lXlo g./well
  • An improved hydrodynamic transfection method "co-injects luciferase siRNA or irrelevant siRNA into a luciferase expression plasmid and introduces it into the liver of adult mice.
  • We are living Liver was taken from animals to monitor luciferase expression, and it was found that the expression level was dependent on the amount of reporter plasmid (results not shown).
  • RNA polymerase III promoter can express functional small hairpin RNAs (shRNAs) from DNA templates in vivo. They are effective because of their suppression and siRNAs.
  • shRNAs small hairpin RNAs
  • An empty shRNA expression vector had no effect (results not shown); the gene was blocked when the shRNA was inserted backward Silencing because it alters the termination of RNA polymerase III and produces shRNAs of inappropriate structure.
  • RNAi will be used in the functional genome or to identify targets for designed drugs. It is a more promising system than knockout mice, because it does not require time-consuming crosses to make groups of genes lose function simultaneously.
  • Gene therapy currently relies on ectopic expression of protein of origin; however, by silencing disease-related genes with DNA vectors that mediate shRNAs expression, RNAi technology finally complements this method of gaining function.
  • Our method of RNAi introduction is also suitable for clinical development of viral and non-viral gene transfer vectors.

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Abstract

La présente invention concerne un vecteur recombinant de virus adéno-associé contenant un néclotide à interférence ARN ARN-i, grâce auquel le gène du nucléotide de l'ARN-i peut être transféré et copié. De cette façon, le nucléotide à interférence ARN peut être doté d'une fonction biologique. Plus spécialement, dans cette invention, nous construisons un vecteur exprimant une boucle en épingle à cheveux courte, inhibant l'expression de la luciférinase dans les cellules BHK-21. Les différentes opérations étant l'utilisation d'ADN génomique humain comme gabarit, la conception d'amorce, le promoteur d'ARN U6sn est produit par amplification en chaîne par polymérase. Ce promoteur est ligaturé avec des répétitions inverses de la séquence de luciférinase 31bp comportant un intervalle 9bp et un psNAV de plasmide AAV. Cela permet de construire l'ARN-i de la luciférinase exprimant le pSNAV/U6/Luc plasmidique qui est cotransfecté dans la cellule BHK-21 avec le pMAMneoLuc plasmidique. Après transfection des pSNAV/U6/Luc et pMAMneoLuc dans les cellules à luciférinase, on détecte l'effet inhibiteur sur l'expression de la luciférinase. Le résultat fait apparaître que le rapport d'inhibition est de 50% dans le précédent et de 70% dans l'actuel, ce qii confirme que la luciférinase contenant des boucles à courtes épingles à cheveux peut inhiber avec succès l'expression de la luciférinase dans les cellules BHK-21.
PCT/CN2003/000685 2002-08-16 2003-08-18 Vecteur de virus adeno-associe inhibant le fonctionnement du gene par le rnai WO2004016780A1 (fr)

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CNA021257620A CN1475573A (zh) 2002-08-16 2002-08-16 利用rna干扰现象介导功能基因下调的腺相关病毒载体
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CN100340667C (zh) * 2004-09-09 2007-10-03 广东省人民医院 用于rna干扰研究的表达载体
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CN105754997B (zh) * 2016-03-30 2019-02-26 华中农业大学 狂犬病毒drv-ah08株的先导rna及其在制备预防与治疗狂犬病毒的药品中的应用

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MORRIS JAMES C. ET AL.: "Glycolysis modulates trypanomosome glycoprotein expression as revealed by an RNAi library", EMBO EUROPEAN MOLECULAR BIOLOGY ORGANIZATION JOURNAL, vol. 21, no. 17, September 2002 (2002-09-01), pages 4429 - 4438, XP002972637, DOI: doi:10.1093/emboj/cdf474 *

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