WO2006058489A1 - Virus adéno-associé recombinant exprimant le gène antisens humain cyp2j2 et méthodes de préparation dudit virus - Google Patents

Virus adéno-associé recombinant exprimant le gène antisens humain cyp2j2 et méthodes de préparation dudit virus Download PDF

Info

Publication number
WO2006058489A1
WO2006058489A1 PCT/CN2005/002040 CN2005002040W WO2006058489A1 WO 2006058489 A1 WO2006058489 A1 WO 2006058489A1 CN 2005002040 W CN2005002040 W CN 2005002040W WO 2006058489 A1 WO2006058489 A1 WO 2006058489A1
Authority
WO
WIPO (PCT)
Prior art keywords
associated virus
gene
cyp2j2
human
recombinant adeno
Prior art date
Application number
PCT/CN2005/002040
Other languages
English (en)
French (fr)
Inventor
Daowen Wang
Jiangang Jiang
Xiao Xiao
Original Assignee
Tongji Hospital, Tongji Medical College, Huazhong University Of Science And Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tongji Hospital, Tongji Medical College, Huazhong University Of Science And Technology filed Critical Tongji Hospital, Tongji Medical College, Huazhong University Of Science And Technology
Priority to US11/720,035 priority Critical patent/US8273344B2/en
Publication of WO2006058489A1 publication Critical patent/WO2006058489A1/zh

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N15/1137—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 against enzymes
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • 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
    • 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
    • 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
    • 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
    • C12N2750/14132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • 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
    • 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 relates to a method for constructing and preparing recombinant adeno-associated virus recombinant (rAAV-anti2J2) capable of expressing human CYP2J2 antisense gene, and more particularly to cloning of human CYP2J2 gene and recombinant adeno-associated virus recombination containing antisense CYP2J2 gene
  • CYP cytochrome P450
  • AA arachidonic acid
  • Metabolic pathways, biological and pathophysiological roles in humans have become hot topics of current research.
  • the AA-CYP oxidase metabolic pathway metabolizes arachidonic acid into four different Epoxyeicosatiienoic Acids (EETs) (5,6-, 8,9-, 11,12-, 14,15-EETs).
  • EETs Epoxyeicosatiienoic Acids
  • CYP oxidases include 2C and 2J.
  • Tumor is a frequently-occurring disease and common disease in modern people. It has a poor prognosis and high mortality. It has been the leading cause of death in China and the world. It is a multi-gene, multi-step, multi-stage chronic disease. At present, the disease cannot achieve good therapeutic effects with general drugs, and with the development of molecular biology theory and technology, the emergence of gene therapy, let us see a new dawn of tumor treatment. Gene therapy has become an important pharmaceutical industry in the 21st century, and there are still many shortcomings to be overcome, especially regarding the choice of gene therapy vectors.
  • the present inventors have succeeded in inserting the CYP2J2 cDNA into the eukaryotic expression vector pXXUFi to construct the recombinant plasmid pXXUF]-anti2J2.
  • pXX 2 , pXX 6 and pXXUFi-antiS were transfected into 293 cells by calcium phosphate co-transfection to prepare recombinant adeno-associated virus capable of expressing human antisense CYP2J2 gene. (rAAV-anti2J2), titer was determined by heparin column purification and dot blot hybridization.
  • the recombinant adeno-associated virus prepared by packaging was transfected into different types of human tumor cell lines. It has been proved that transfection of recombinant adeno-associated virus containing antisense CYP2J2 can significantly inhibit the proliferation and migration of tumor cells and promote the apoptosis of tumor cells. After death, the cell line transfected with rAAV-anti2J2 was introduced into the skin of nude mice. It was found that rAAV-anti2J2 significantly inhibited the growth of tumor in vivo, and the selective inhibitor of CYP2J2 also played the same role. This provides a new way of thinking and direction for the development of new drugs for cancer treatment.
  • a first object of the present invention is to provide a recombinant adeno-associated virus containing the antisense human CYP2J2 gene.
  • a second object of the present invention is to provide a process for the preparation and preparation of a recombinant adeno-associated virus containing the antisense human CYP2J2 gene.
  • a third object of the present invention is to provide an experimental method and result for inhibiting tumor cell proliferation and treating tumors using CYP oxidase selective inhibitor and EET blocker.
  • a fourth object of the present invention is to provide an experimental method and result for treating a tumor using a recombinant adeno-associated virus containing the antisense human CYP2J2 gene.
  • a recombinant adeno-associated virus expressing a human CYP2J2 antisense gene and a preparation method thereof are designed by the inventors, and the recombinant adeno-associated virus provided by the present invention is 1509 bp long from human CYP2J2 cDNA, and contains 503 amino acid protein, human CYP2J2 cDNA was cloned from human leukocyte DNA by PCR, recombinant adeno-associated virus containing antisense gene was prepared by three plasmid calcium phosphate co-transfection, and purified recombinant adeno-associated virus was prepared.
  • Transfection of different types of human tumor cell lines can significantly inhibit the proliferation and migration of various tumor cells, promote the apoptosis of tumor cells, and inhibit the growth and metastasis of tumor tumors in vivo. Therefore, it was confirmed that a selective inhibitor of CYP2J2 and a recombinant adeno-associated virus expressing the antisense CYP2J2 gene are potential drugs for tumor therapy.
  • the recombinant adeno-associated virus of the present invention comprises three kinds of plasmids:
  • pXX 2 is a packaging plasmid containing a coding sequence encoding the adeno-associated virus Rep protein and Cap protein. And insert a p5 promoter in the upstream and downstream, so that the expression efficiency can be increased by 15 times, providing the Rep protein necessary for rAAV replication;
  • P XXUF i eukaryotic expression vector, containing CMV promoter, has strong expression efficiency, contains a polyclonal Notl site, can be linked to different genes of interest, pXXUFi reversely linked human CYP oxidase gene
  • the vector contains terminal repeats (ITRs) necessary for expression of the rAAV transgene, and is responsible for viral replication and coating of the viral envelope and carrying the gene of interest;
  • pXX 6 is a helper plasmid, deletes the pathogenic gene sequence of adenovirus, retains the adenovirus E1A, E2A and VAi RNA genes, and the expressed protein can play a supporting role to stimulate the transcription and translation of rAAV gene, ensuring rAAV production;
  • XX 2 plasmid - a coding sequence for Rep and Cap proteins necessary for packaging of adeno-associated viruses, and a pXX 6 plasmid - providing adenoviruses E A, E2A and VAI necessary for stimulation of adeno-associated virus replication and transcription RNA gene;
  • the adeno-associated virus lacks the cap protein and rep protein coding region, and inserts the human CYP2J2 antisense gene to form a recombinant plasmid pXXUF!-antiSJS;
  • the purified virus was transfected into tumor cells, and its expression and biological activity were examined.
  • the recombinant adeno-associated virus is artificially cut, modified and processed by a molecular biological method in a natural state, and then packaged and replicated by molecular biological methods and purified. It includes a recombinant adeno-associated virus expressing the human CYP2J2 antisense gene and a pharmaceutically acceptable carrier or excipient.
  • the recombinant adeno-associated virus expressing the human CYP2J2 antisense gene can selectively block the expression of CYP2J2 gene in tumor tissues, inhibit the synthesis of epi-epoxicosatrienoic acid and treat malignant tumors, and select CYP table.
  • Oxidase-selective inhibitors and CYP-epoxidases catabolize the oxidation of arachidonic acid to produce epi-epoxidized eicosyltrienoic acid antagonists. These and similar chemicals are artificially combined and selectively inhibited.
  • CYP oxidase activity or blocking the action of epoxidized ecamosatrienoic acid can control tumor proliferation without toxic effects on cells.
  • the recombinant adeno-associated virus expressing the human CYP212 antisense gene of the present invention has been preserved at the China Center for Canonical Conservation in Wuhan University, and the date of deposit: June 30, 2004, preservation number: CCTCCNO: V200411 Classification: Recombinant adeno-associated virus expressing the human cytochrome P4502J2 antisense gene.
  • CCTCCNO V200411 Classification: Recombinant adeno-associated virus expressing the human cytochrome P4502J2 antisense gene.
  • the invention is described in further detail:
  • the inventors In order to obtain the human CYP2J2 gene, the inventors first designed a specific primer for amplifying the CYP2J2 cDNA fragment according to the CYP2J2 gene sequence reported by GeneBank, and extracted genomic DNA from human leukocytes by phenol-chloroform extraction and used it as a template.
  • PCR kit was purchased from TaKaRa, Japan). The PCR machine was purchased from TECONE, UK.
  • the product was reversely ligated with pXXUFi to form a recombinant plasmid pXXUFrantiS, which was introduced into the host cell, Escherichia coli, to obtain a positive clone.
  • Recombinant adeno-associated virus packaging requires three plasmids, namely (1) ⁇ 2: a packaging plasmid containing a coding sequence encoding the adeno-associated virus Rep protein and Cap protein. And insert a p5 in the upstream and downstream The promoter can increase the expression efficiency by 15 times; (2) pXX 6: is a helper plasmid, deletes the disease-causing gene sequence of adenovirus, retains the adenovirus E1A, E2A and VA1 RNA genes, and the proteins expressed by them can play an auxiliary role. Function to stimulate transcription and translation of AAV gene to ensure AAV production; (3)
  • PXXUF,-anti2J2 The eukaryotic expression vector of CYP2J2 gene is reversely linked, and the CMV promoter is used, which has strong expression efficiency. This vector contains the terminal repeats necessary for rAAV transgene expression
  • ITRs responsible for viral replication and viral envelopes and carrying the gene of interest.
  • ITRs responsible for viral replication and viral envelopes and carrying the gene of interest.
  • pXX 2 , pXX 6 , pXXUF!-anti2J2 was transferred to 293 cells by calcium phosphate transfection. 48-72 hours after transfection, 293 cells were harvested, and the cells were repeatedly thawed three times. The virus was released into the supernatant, purified by heparin column, and the titer was determined by dot blot hybridization to obtain the antisense human 2J2 gene. Recombinant adeno-associated virus.
  • rAAV-anti2J2 was transfected into human tongue squamous cell carcinoma cell line Tca-8113, and then transplanted into nude mice, partially mimicking the growth process of tumor cells in vivo, measuring tumor size, and plotting tumor volume growth curve. It was proved that rAAV-anti2J2 transfection can significantly inhibit tumor growth.
  • microvascular density measurements of tumor tissues revealed that rAAV-anti2J2 transfection significantly reduced microvessel density in the tumor.
  • rAAV-anti2J2 transfection can significantly reduce the migration ability of tumor cells. Promotes apoptosis of tumor cells.
  • AV-anti2J2 and CYP oxidase inhibitor (17-ODYA) have the effects of inhibiting tumor proliferation and metastasis and promoting tumor cell apoptosis, and can be used for tumor treatment.
  • the recombinant adeno-associated virus (rAAV-anti2J2) expressing human CYP2J2 antisense gene was successfully constructed and expressed in vivo for a long time. It blocks the transcription and translation of CYP2J2 gene at the gene level, and reduces the expression of CYP2J2. , more thorough.
  • rAAV-anti2J2 recombinant adeno-associated virus
  • CYP oxidase inhibitors expressing the human CYP2J2 antisense gene
  • the advantage of the present invention is that the recombinant adeno-associated virus vector researched and developed overcomes the shortcomings that other gene expression vectors are difficult to overcome, and can carry the target gene to transfect both mitotic and non-dividing cells (ie, having a wide range of transgenes). It has no side effects (no immunogenicity), high infection efficiency, can drive long-term expression of the target gene in vivo, and successfully solves the problem of large-scale replication in vitro without adenovirus contamination.
  • Figure 1 shows the sequence of 2J2 (taken from GeneBank) and the light part is the CDNA open reading frame sequence
  • Figure 2 is a diagram showing the plasmid composition of pXXU1 antiS
  • Figure 3 shows the nucleotide sequence and specific partition of the adeno-associated virus
  • Figure 4 is a diagram showing the genomic structure of an adeno-associated virus
  • Figure 5 is a diagram showing the ITR sequence and secondary structure of an adeno-associated virus
  • Figure 6 shows the transcription and translation of adeno-associated virus (1)
  • Figure 7 shows the transcription and translation of adeno-associated virus (2)
  • Figure 8 is a diagram showing the composition of plasmid maps of ⁇ 2 and ⁇ 6 ;
  • Figure 9 is a diagram showing the construction of pXXUF 1 plasmid (Note: This plasmid consists only of the major part of the plasmid. In fact, the two plasmids have been processed into a cyclic structure of 5000-7000 bp, and the rest are not important, so they are not written.) ;
  • Figure 10 is a graph showing that the efficient expression of the CYP-table oxidase gene (rAAV-2J2) after transfection and the antisense gene (rAAV-anti2J2) significantly inhibited the expression of CYP2J2 in tumor cells
  • Fig. 10A CYP oxidase gene (rAAV) -2J2) Efficient expression after transfection and antisense gene (rAAV-anti2J2) significantly inhibited CYP2J2 expression in tumor cells after transfection.
  • A transfected tumor cells with rAAV-2J2 and rAAV-anti2J2, respectively, and implanted into nude mice to form a transplanted tumor.
  • CYP-2J2 Western Blot results After 32 days, the protein extracted from the transplanted tumor tissue was subjected to CYP-2J2 Western Blot results; B, rAAV-2J2 was used respectively.
  • the results of CYP-2J2 Western Blot were directly extracted from the tumor cells after transfection with rAAV-anti2J2. Both of these results indicated that rAAV-2J2 transfection could be efficiently expressed in tumor cells, while rAAV-anti2J2 transfection was very significant. Inhibition or blockade of CYP2J2 expression. This is closely related to the malignant proliferation of tumor caused by CYP2J2 and the malignant proliferation of tumor by rAAV-anti2J2;
  • Figure 11 is a graph showing the effect of CYP oxidase on the proliferation of four tumor cell lines; ' Figure 12 shows the tumor growth curve after transgene;
  • Figure 13 is a graph showing the results of microvascular density of tumor tissue after transfection of a gene
  • Figure 14 is a graph showing the effect of CYP oxidase on tumor cell invasion ability.
  • Figure 15 shows the dose-dependent inhibition of tumor cell growth by the surface oxidase inhibitor 17-ODYA.
  • Figure 16 shows the inhibitory effect of EET blocker 14, 15-EET (100 nmol/L) on tumor growth
  • Figure 17 shows the growth of xenografts by the surface oxidase inhibitor 17-ODYA alone or in combination with 5-fluorouracil. Inhibition.
  • Example 1 Cloning of CYP2J2 cDNA and preparation of P XXUF, -anti2J2 recombinant plasmid
  • the PCR cleavage of the CYP2J2 cDNA was first designed according to the published CYP2J2 gene sequence (Fig. 1): upstream primer: 5 ' - GCCCGGAATTCAAAATGATTCTCAAC-3'; downstream primer: 5 ' - GGCGCACAAGCTTTCAAATAAGAGTATAAC-3' . Synthesized by Wuhan Biosynthetic Company.
  • Genomic DNA was extracted from human leukocytes by phenol-chloroform extraction and PCR was carried out using the template as a template (the kit was purchased from TAKARA, Japan). The PCR instrument was purchased from TECONE, UK. The product was ligated in reverse to pXXUF to form the recombinant plasmid pXXUF r anti2J2. The plasmid map is shown in Figure 2.
  • Example 2 Packaging, recovery and purification of rAAV-anti2J2 recombinant virus
  • Adeno-associated virus is an animal single-stranded DNA virus belonging to the family Parvoviridae, subfamily of parvoviruses, dependent on genus of viruses, natural defects No coating and no pathogenicity.
  • the AAV genome is a linear, single-stranded (ssDNA) molecule containing 4680 nucleotides (sequence as shown in Figure 3), which is characterized by -
  • the genome consists of four open reading frames (ORFs), which are divided into rep, lip, inf and cap regions (see Figure 3, Figure 4).
  • ORFs open reading frames
  • a large ORF located at the left end of the genomic DNA is called a rep region because of a frameshift mutation or a deletion that prevents DNA replication.
  • a large ORF (cap) at the right end encodes three coat proteins.
  • Two other small ORFs are located in the central region of the genomic DNA, namely the inf and lip regions, and the specific functions are still unclear.
  • ITR 145 base end repeat
  • the ITR sequence is folded into a hairpin structure as a DNA replication initiation and packaging recombination.
  • the AAV genome is the only known cis-acting element required for infectious viral particles (see Figure 5).
  • Ad adenovirus
  • Ad E2A gene encodes a single-stranded DNA-binding protein that stimulates transcription initiated by the AAV promoter and promotes AAV.
  • AAV also positively regulates and negatively regulates the expression of its own genes and helper genes.
  • the rep gene of AAV can positively regulate and negatively regulate the transcription initiated by the P5, P19, and P40 promoters.
  • the rep gene product performs a positive regulatory function, which is necessary for the large-scale synthesis of the AAV gene;
  • the rep gene product plays a negative regulatory role.
  • AAV DNA is integrated into the host cell genome in a double-stranded form where it persists in a latent state, forming a latent infection of AAV.
  • the specific site of AAV integration is on the 19ql3.3-19q ter of the human chromosome.
  • Rep gene products can also recognize Binding to a specific integration site, the GGTC sequence, and mediating the recombination of ITR and integration sites. Adenovirus infection can cause it to enter a proliferative infection state.
  • AAV is used as a carrier for gene therapy.
  • AAV is the only eukaryotic cell virus currently known to be able to integrate at a specific chromosomal site in the host genome. This discovery has given rise to new hopes for gene therapy. Compared to non-integrating vectors (such as adenovirus-based vectors), AAV vectors not only enable more stable and stable expression of transgenes, but also reduce theoretically due to random integration of transgenes. Insert a mutation probability.
  • the set of vector systems we use is a series of cuts and modifications of natural adeno-associated viruses and adenoviruses using molecular biology methods that retain the essential parts of the replication and transcription of adeno-associated viruses, as well as the glands.
  • the late gene-assisted components of the virus have been deleted, which can achieve long-term stable latent infection of the virus and avoid the risk of adenovirus, and the expression of late genes of the adenovirus can ensure the expression efficiency of the cloned gene.
  • the composition of this carrier system is as follows -
  • pXX 2 (Fig. 8) retains the cap gene and rep gene of AAV (when pACG-2 is obtained at this time), and inserts a p5 promoter in the downstream region (cut in the original region with Xbal and Pstl, and then Connect to the appropriate location of pACG-2).
  • PXX 6 (Fig. 8) An 8 kb fragment was obtained with Pmel and Sgfl on the adenovirus engineered plasmid ⁇ (the El, E3 gene and packaging signals were cleaved) (pXX 5 was obtained), while pXX 6 was On PXX 5 , Clal and Sail were used to cut a clone into the pBS, leaving only the E2A, E4 and VA coding regions.
  • pXXUF (Fig. 9): The plasmid pXXUF used in rAAV packaging contains the human gfp gene driven by the CMV promoter (arrow), which deletes all the genes encoding the adeno-associated virus and then imports them from the Notl site. In order to facilitate gene cloning and expression, it is processed into a loop.
  • 293 cells human kidney mother embryo tumor cell line
  • trypsin high glucose DMEM medium
  • newborn calf serum purchased from Gibco.
  • the calcium phosphate transfection method is carried out according to a molar ratio of 1:1:1 (mass ratio: 1.7:3.8:1). Into 293 cells.
  • pXX 2, pXX 6, pXXUFi- antiS plasmid was added a 250ml sterile flask, followed by addition of a solution of 2.5MCaCl 2 (160 ⁇ 1 / dish) and sterile deionized water and finally 2 X BBS (pH6. 95, 1.6 ml / plate), shake the mixture dropwise to form smaller calcium phosphate particles. Incubate 30 ⁇ at room temperature. Immediately abandon the medium in the 293 cell culture dish, add 3ml DNA-calcium phosphate mixture, culture at 35 ° C, 3% CO 2 for 16 ⁇ 24 hours, then change the solution, transfer to 37 ° C, continue to culture 5% CO 2 ⁇ 48 hours. Turn All medium was aspirated 48-72 hours after dyeing, a small amount of PBS buffer was added, and 293 cells in the dish were scraped off, -80
  • the method is as follows: The collected virus-containing 293 cells are repeatedly thawed three times, and O.lmg of DNase I and O.lmg of RNase A are added, and 37 °C is incubated.
  • the isotope-labeled nucleotide ⁇ -P 32 was purchased from Beijing Yahui Company. P XXUF was cut with Not I, and the CYP2J2 fragment in -anti2J2 was used as a probe for the known sequence. The CYP2J2 fragment was labeled by random primer method and purified (kit was purchased from QIAGEN, method as described).
  • the spot hybridization device is equipped, and a 0.45 ⁇ nylon membrane is placed according to the size of the device.
  • the virus sample and the standard sample to be tested are respectively loaded according to the volume gradient and the molecular gradient.
  • the membrane 8CTC is dry-baked for 2 hours, after 42 Prehybridization was carried out for 1 hour at °C (prehybridization was purchased from INTERGEN).
  • the heat-treated labeled probe was further mixed and hybridized at 45 ° C for 12-16 hours. After washing the film - autoradiography at -80 °C for 2-3 days.
  • the prepared rAAV titer reaches 1 X lOUp.f.u. and can be used in animal experiments.
  • transfection of rAAV-CYP2J2 and rAAV-CYPF87V significantly promoted the proliferation of tumor cells, and the number of cell proliferation was 2 to 3 times that of the basal state transfected with rAAV-GFP and untransfected cells (PO.01); After transfection of the antisense CYP oxidase gene (rAAV-anti2J2) and the addition of the CYP oxidase inhibitor 17-ODYA, cell proliferation was significantly inhibited, which was less than 50% of the basal state (P ⁇ 0.01). See Figure 11.
  • microvessel density (MVD) count analysis After 32 days of inoculation, the animals were sacrificed, and the paraffin sections were excised to prepare microvessel density (MVD) count analysis. Microscopically, microvessels are mainly distributed in the interstitial and peripheral regions of the tumor tissue, while there are few microvascular distributions in the tumor tissue. The microvascular vessels in the rAAV-CYP2J2 and rAAV-CYPF87V groups were increased, and the vascular density was larger.
  • the MVD in the high power field was 76.8 ⁇ 9.1 and 70.2 ⁇ 7.8, respectively, which was significantly different from the control group (42.8 ⁇ 6.4) (P ⁇ 0.01), while the transfected rAAV-anti2J2 group (23.6 ⁇ 7.3) was significantly lower in microvessel density compared with the control group and the transfected i'AAV-CYP2J2 group (P ⁇ 0.05 and corpse ⁇ 0.01). See Figure 13.
  • rAAV-anti2J2 virus and CYP oxidase inhibitor (17-ODYA) have the effect of inhibiting tumor proliferation and metastasis and can be used for tumor treatment.
  • the recombinant adeno-associated virus (rAAV-anti2J2) expressing human CYP2J2 antisense gene was successfully constructed and expressed in vivo for a long time. It blocks the transcription and translation of CYP2J2 gene at the gene level, and reduces the expression of CYP2J2. , more thorough.
  • rAAV-anti2J2 recombinant adeno-associated virus
  • CYP oxidase inhibitors which are believed to express the human CYP2J2 antisense gene, it will be extremely important in the treatment of diseases such as tumors that are seriously harmful to human health. The role.
  • EETs and oxidase genes have anti-TNF-oi-induced apoptosis in tumor cells; 3 TNF analysis by flow cytometry after PI staining and Annexin V-FITC/PI double staining, respectively - ⁇ -induced total cell apoptosis rate and early apoptotic rate.
  • oxidase and EETs not only significantly promote tumor growth, but also protect tumor cells by inhibiting TNF-CC-induced apoptosis. Therefore, according to these results, CYP-table oxidase inhibitor or transfection of rAVV-anti2J2 can be used to promote tumor apoptosis for the purpose of treating tumors.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Virology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Description

表达人类 CYP2J2反义基因的重组腺相关病毒及其制备方法 发明领域
本发明涉及能表达人类 CYP2J2 反义基因的重组腺相关病毒重组体 (rAAV-anti2J2) 的构建和制备方法， 更具体地说涉及人类 CYP2J2基因的克隆 及含反义 CYP2J2基因的重组腺相关病毒重组体的包装制备方法，和该重组腺相 关病毒重组体以及 CYP表气化酶选择性抑制剂及二十烷基三烯酸阻断剂治疗肿 瘤的药学用途。
背景技术
随着人们对细胞色素 P450 (CYP) 氧化酶系广泛研究和认识， 其在机体生 理和病理生理方面所起的作用日益受到重视， 尤其是 20年前发现的花生四烯酸 (AA)的 CYP代谢途径， 在人体中的生物学及病理生理学作用成为目前研究的 热点。 AA-CYP表氧化酶代谢途径能将花生四烯酸代谢成 4种不同的表氧化二十 碳三烯酸 (Epoxyeicosatiienoic Acids, EETs) (5,6-, 8,9-， 11,12-, 14,15-EETs)。 CYP 表氧化酶包括 2C和 2J两类， 已有研究显示， 2C在生物体内广泛分布， 尤其在 肝脏中表达最丰富， 在血管中亦存在表达； 目前已发现 2J 的 6个克隆， 其中在 人类仅发现了 2J2, 其在心脏和血管内皮细胞表达最丰富。 我们和国外学者已有 研究发现 EETs在调节心血管系统稳态中发挥着重要作用， 包括调节血压、 对心 脏和血管内皮细胞的保护及抗凋亡作用。然而我们的研究发现表氧化酶基因在人 类肿瘤组织中选择高表达， 并能促进肿瘤细胞的增殖转移和抗肿瘤细胞的凋亡。 这一发现对肿瘤的治疗具有极其积极的意义。 肿瘤是现代人群的多发病及常见 病， 预后差、 死亡率高， 在我国乃至世界一直是主要的致死原因， 它是一种多基 因、 多步骤、 多阶段调控的慢性疾病。 目前该疾病用一般药物不能取得良好的治 疗效果， 而随着分子生物学理论和技术的发展， 基因治疗的出现， 让我们看到肿 瘤治疗新的曙光。 基因治疗已成为 21世纪重要的医药产业， 尚有很多有待克服 的缺点， 尤其是关于基因治疗载体的选择。
为了能使肿瘤的基因治疗真正实现， 我们将反义人类 CYP2J2 cDNA与重组 腺相关病毒载体重组构建， 并经检测已获得了可以满足治疗要求的高滴度病毒， 在动物实验中也已证实了其可以有抑制肿瘤生长作用，从而有希望真正满足临床 治疗的需要。 此外研究了 AA-CYP表氧化酶的抑制剂抑制恶性肿瘤增殖的作用 及其机制。 阐述了 AA-CYP表氧化酶在人类肿瘤中选择性高表达及其抑制剂抑 制恶性肿瘤增殖的药学用途。
发明内容
本发明人已经成功将 CYP2J2 cDNA反向插入真核表达载体 pXXUFi中构 成重组质粒 pXXUF】-anti2J2。 之后， 将 pXX2、 pXX6 、 pXXUFi-antiS 用钙磷 共转染法转入 293细胞包装制备能表达人反义 CYP2J2基因的重组腺相关病毒 (rAAV-anti2J2), 采用肝素柱纯化， 斑点杂交法测定滴度。另外， 将包装制备的 重组腺相关病毒转染不同种类的人肿瘤细胞系， 经实践证明含反义 CYP2J2的重 组腺相关病毒转染能非常显著地抑制肿瘤细胞增殖和迁移， 促进肿瘤细胞的凋 亡， 而将转染了 rAAV-anti2J2的细胞系导入裸鼠皮下发现 rAAV-anti2J2能显著 抑制体内瘤体的生长， 此外 CYP2J2的选择性抑制剂亦起到了同样的作用。 这为 开发肿瘤治疗新药提供了全新的思路和方向。
所以， 本发明的第一个目的是提供含有反义人 CYP2J2基因的重组腺相关病 毒。 ,
本发明的第二个目的是提供含有反义人 CYP2J2基因的重组腺相关病毒的包 装和制备过程。
本发明的第三个目的是提供利用 CYP表氧化酶选择性抑制剂和 EET阻断剂 抑制肿瘤细胞增殖及其治疗肿瘤的实验方法和结果。
本发明的第四个目的是提供利用含反义人 CYP2J2基因的重组腺相关病毒治 疗肿瘤的实验方法和结果。
为了实现本发明的目的， 发明人设计的一种表达人类 CYP2J2反义基因的重 组腺相关病毒及其制备方法， 本发明所提供的该重组腺相关病毒， 是从人类 CYP2J2 cDNA长 1509bp，编码含 503个氨基酸的蛋白质，利用 PCR法从人类白 细胞 DNA中克隆人类 CYP2J2 cDNA,通过三种质粒磷酸钙共转染法包装制备含 反义目的基因的重组腺相关病毒并纯化，制备的重组腺相关病毒转染不同种类的 人肿瘤细胞系，它能明显抑制各种肿瘤细胞的增殖和迁移、促进肿瘤细胞的凋亡， 抑制在体肿瘤瘤体的生长和转移。 因此， 确定， CYP2J2的选择性抑制剂和表达 反义 CYP2J2基因的重组腺相关病毒是肿瘤治疗的潜在药物。
本发明重组腺相关病毒包括的三种质粒：
( 1 ) pXX2: 为包装质粒， 含有编码腺相关病毒 Rep蛋白和 Cap蛋白的编码 序列。 并在上游和下游各插入一个 p5启动子， 使表达效率可以提高 15倍， 提供 rAAV复制所必须的 Rep蛋白；
(2) PXXUFi : 真核表达载体， 含 CMV启动子， 有较强的表达效率， 其含 有一多克隆 Notl位点， 可以连接不同的目的基因， pXXUFi反向连接人 CYP表 氧化酶基因， 该载体含有 rAAV转基因表达所必须的末端重复序列 （ITRs), 负 责病毒的复制和病毒外壳的包被并携带目的基因；
(3 ) pXX6: 为辅助质粒， 删除了腺病毒的致病基因序列， 保留了腺病毒 E1A、 E2A和 VAi RNA基因， 所表达的蛋白质可发挥辅助作用以刺激 rAAV基 因的转录和翻译， 保证 rAAV产量；
(4) 以上三种质粒经磷酸钙沉淀法共转染 293细胞， 即可在 293细胞内形 成高滴度的含目的基因的 rAAV颗粒。
本发明重组腺相关病毒的制备， 其步骤：
( 1 ) XX2质粒——提供腺相关病毒包装所必需的 Rep蛋白和 Cap蛋白的编 码序列，和 pXX6质粒——提供刺激腺相关病毒复制和转录所必需的腺病毒 El A、 E2A和 VAI RNA基因； -
(2) 含腺相关病毒基因组的真核表达载体 pXXUF^ 在该真核载体中腺相 关病毒缺失 cap蛋白和 rep蛋白编码区， 并插入人类 CYP2J2反义基因， 形成重 组质粒 pXXUF!-antiSJS;
(3 ) 步骤 1 ) 中的 pXX2、 pXX6和步骤 2) 中的 pXXUF!-an^ 通过磷酸 钙沉淀法共转染 293细胞， 适时回收， 纯化、 斑点杂交法检测其病毒滴度；
(4) 步骤 3 ) 中回收纯化的病毒转染肿瘤细胞， 检测其表达及生物学活性。 该重组腺相关病毒由天然状态的腺相关病毒经分子生物学方法人工剪切、修 饰和加工， 再经分子生物学方法包装复制， 并经纯化而成。 它包括有表达人类 CYP2J2反义基因的重组腺相关病毒和药学上所接受的载 体或赋形剂。
本发明所述的一种表达人类 CYP2J2反义基因的重组腺相关病毒， 能选择性 阻断肿瘤组织中 CYP2J2基因表达，抑制表氧化二十烷三烯酸合成而治疗恶性肿 瘤，其选用 CYP表氧化酶选择性抑制剂和 CYP表氧化酶代谢花生四烯酸所形成 的表氧化产生表氧化二十烷三烯酸的拮抗剂，这些及类似的化学制剂是人工化合 而成并具有选择性抑制 CYP 表氧化酶系活性或阻断表氧化二十烷三烯酸的作 用， 能够控制肿瘤增殖， 而对细胞并无毒性作用。
本发明所述的一种表达人类 CYP212反义基因的重组腺相关病毒，已保藏于 武汉大学内中国典型物保护中心， 保藏日期： 2004年 6月 30 日， 保藏编号： CCTCCNO: V200411分类命名： 表达人类细胞色素 P4502J2反义基因的重组腺 相关病毒。 本发明进一步详细描述：
为了获得人 CYP2J2基因， 本发明人首先根据 GeneBank报道的 CYP2J2基 因序列设计了扩增 CYP2J2 cDNA片段的特异性引物，用酚-氯仿抽提的方法从人 白细胞中提取基因组 DNA并以其为模板进行 PCR (试剂盒购自日本 TaKaRa公 司）。 PCR仪购自英国 TECONE公司。并将产物与 pXXUFi反向相连构成重组质 粒 pXXUFrantiS , 导入寄主细胞即大肠杆菌， 获得阳性克隆。
重组腺相关病毒包装需要三种质粒， 即 （1 ) ρΧΧ2 : 为包装质粒， 含有编码 腺相关病毒 Rep蛋白和 Cap蛋白的编码序列。 并在上游和下游各插入一个 p5启 动子， 使表达效率可以提高 15倍； （2) pXX6: 为辅助质粒， 删除了腺病毒的致 病基因序列， 保留了腺病毒 E1A、 E2A和 VA1 RNA基因， 它们表达的蛋白质可 发挥辅助作用以刺激 AAV基因的转录和翻译， 保证 AAV的产量； （3 )
PXXUF,-anti2J2: 反向连接人 CYP2J2基因的真核表达载体， 所用的是 CMV启 动子， 有较强的表达效率。 该载体含有 rAAV转基因表达所必须的末端重复序列
(ITRs), 负责病毒的复制和病毒外壳的包被并携带目的基因。 我们按摩尔比 1:1:1将 pXX2、 pXX6、 pXXUF!-anti2J2用钙磷转染法转入 293细胞。转染后 48-72 小时后， 收获 293细胞， 反复冻溶细胞 3次， 病毒即释放入上清液中， 釆用肝素 柱纯化， 斑点杂交法测定滴度， 即获得含反义人 2J2基因的重组腺相关病毒。
然后为了证实 CYP表氧化酶在肿瘤发生发展中的作用及其选择性抑制剂和 含反义 2J2基因的重组腺相关病毒对肿瘤的治疗作用。 首先研究开发 CYP表氧 化酶在人类肿瘤组织中选择性高表达，并证明 rAAV-anti2J2能显著降低肿瘤细胞 2J2的表达。而转染反义 CYP表氧化酶基因（rAAV-anti2J2)和加用 CYP表氧化 酶抑制剂 17-ODYA可显著抑制肿瘤细胞增殖， 为基础状态下的 50%以下
(P<0.01 ); 而转染 rAAV-CYP2J2能明显促进肿瘤细胞的增殖， 细胞增殖数是基 础状态即转染 rAAV-GFP和未转染细胞的 2〜3倍 (PO.01 )。同样将 rAAV-anti2J2 转染人舌鳞癌细胞株 Tca-8113， 然后移植到裸鼠皮下， 部分模拟肿瘤细胞在体内 的生长过程， 测量肿瘤大小， 绘制肿瘤体积生长曲线。证明 rAAV-anti2J2转染能 明显抑制肿瘤的生长。
此外， 对肿瘤组织微血管密度测定发现， rAAV-anti2J2转染能显著降低瘤体 内微血管密度。
同样 rAAV-anti2J2转染还能显著降低肿瘤细胞的迁移能力。促进肿瘤细胞的 凋亡。
上述结果显示 AV-anti2J2及 CYP表氧化酶抑制剂 （ 17-ODYA) 具有抑制 肿瘤增殖转移、 促进肿瘤细胞凋亡的作用， 可用于肿瘤治疗。 表达人类 CYP2J2 反义基因的重组腺相关病毒 （rAAV-anti2J2) 的构建成功， 在生物体内可长期高 效表达， 从基因水平上阻断 CYP2J2基因的转录与翻译， 使 CYP2J2表达减少， 其作用更强， 更为彻底。 将其与表达人类 CYP2J2 反义基因的重组腺相关病毒 (rAAV-anti2J2)和 CYP表氧化酶抑制剂用于临床后，必将在肿瘤等严重危害人 类健康的疾病治疗中发挥极其主要的作用。
本发明的优越性在于，其研究开发的重组腺相关病毒载体克服了其他基因表 达载体所难以克服的缺点， 它可以携带目的基因转染分裂期和非分裂期细胞（即 具有广泛的转基因范围）、 无副作用 （无免疫源性）、 感染效率高、 能驱动目的基 因在体内长期表达， 而且成功地解决了无腺病毒污染的体外大量复制问题。 附图说明
从下面给出附图， 结合本发明的上面和其他目的和特征将变得明了。
图 1显示了 2J2 的序列 （取自 GeneBank), 浅色部分为 CDNA开放阅读框 序列；
图 2是显示 pXXUl antiS 的质粒构成；
图 3显示了腺相关病毒的核苷酸序列及具体分区；
图 4是显示腺相关病毒的基因组构造图；
图 5是显示腺相关病毒的 ITR序列及二级结构图；
图 6是显示腺相关病毒的转录和翻译 （一） ；
图 7是显示腺相关病毒的转录和翻译 （二） ；
图 8是显示 ρΧΧ2、 ρΧΧ6质粒图谱构成；
图 9是显示 pXXUF1质粒构成 （注： 该质粒构成图只显示质粒的主要部分， 实际上这两种质粒已加工为 5000-7000bp的环状结构， 其余部分无重要性， 故不 写入） ；
图 10是显示 CYP表氧化酶基因（rAAV-2J2)转染后的有效表达和反义基因 (rAAV-anti2J2)转染后显著抑制肿瘤细胞 CYP2J2表达，其中图 10A. CYP表氧 化酶基因 （rAAV-2J2) 转染后的有效表达和反义基因 （rAAV-anti2J2) 转染后显 著抑制肿瘤细胞 CYP2J2表达。 A, 分别用 rAAV-2J2和 rAAV-anti2J2转染肿瘤 细胞后种植于裸鼠皮下形成移植瘤， 32 天后从移植瘤组织中提取的蛋白进行 CYP-2J2 Western Blot结果； B， 分别用 rAAV-2J2和 rAAV-anti2J2转染肿瘤细胞 后直接提取的蛋白进行 CYP-2J2 Western Blot结果，这两个结果均显示 rAAV-2J2 转染能在肿瘤细胞中有效表达， 而 rAAV-anti2J2 转染能非常显著地抑制或阻断 CYP2J2表达。 这与 CYP2J2引起肿瘤恶性增殖和 rAAV-anti2J2抑制肿瘤恶性增 殖密切相关；
图 11是显示 CYP表氧化酶对四肿瘤细胞系增殖的影响； ' 图 12是显示转基因后肿瘤生长曲线；
图 13是显示转染基因后肿瘤组织微血管密度结果；
图 14是显示 CYP表氧化酶对肿瘤细胞侵袭能力的影响。
图 15显示表氧化酶抑制剂 17-ODYA对肿瘤细胞生长的剂量依赖性抑制作 用。
图 16显示 EET阻断剂 14， 15-EET ( 100nmol/L) 对肿瘤生长的抑制作用； 图 17显示表氧化酶抑制剂 17-ODYA单用非联合使用 5-氟脲嘧啶对移植瘤 生长的抑制作用。
具体实施方式
实施例 1CYP2J2 cDNA的克隆及 PXXUF,-anti2J2重组质粒的制备 为了克隆人 CYP2J2 cDNA根据公开的 CYP2J2基因序列 (图 1)首先设计了可 扩 增 CYP2J2 cDNA 的 PCR 弓 I 物 ： 上 游 引 物 ： 5 ' — GCCCGGAATTCAAAATGATTCTCAAC 一 3 ' ； 下 游 引 物 ： 5 ' — GGCGCACAAGCTTTCAAATAAGAGTATAAC— 3'。 由武汉生物合成公司合成。 用酚-氯仿抽提的方法从人白细胞中提取基因组 DNA并以其为模板进行 PCR (试 剂盒购自日本 TAKARA公司）。 PCR仪购自英国 TECONE公司。 并将产物与 pXXUF,反向相连， 构成重组质粒 pXXUFranti2J2。 质粒图如图 2。
实施例 2 rAAV-anti2J2重组病毒的包装、 回收与纯化
―、 天然腺相关病毒 （AAV) 及重组腺相关病毒 （rAAV) 载体的特点： 腺相关病毒是一种动物单链 DNA病毒， 它属于细小病毒科， 细小病毒 亚科， 依赖病毒属， 自然缺陷、 无包被和无致病原性。
1、 AAV基因组是一个线性、 单链 (ssDNA)分子， 含 4680个核苷酸 (序列如 图 3)， 其特点在于-
1 )其基因组由 4个开放阅读框 （ORF) 组成， 分称 rep区、 lip区、 inf区和 cap区 （见图 3、 图 4)。 位于基因组 DNA左端的一个大 ORF由于移码突变或缺 失会阻止 DNA复制， 因而被称为 rep区，。 右端的一个大 ORF (cap) 编码合成 3种外壳蛋白。 另有两个小 ORF位于基因组 DNA的中央区， 即 inf区和 lip区， 具体功能尚不清。
2) 在分子单链每个末端含有一个 145个碱基末端重复序列 (ITR) (见图 3 )。 ITR序列折叠成发卡结构作为 DNA复制起始和包装重组 AAV基因组为感染性的 病毒颗粒所需的唯一已知顺式作用元件 （见图 5)。
3)腺相关病毒的复制和转录调节相当复杂， 按有无辅助病毒的共转染分， 有 增殖性和潜伏性两种表达方式 （如图 6) 。
在 AAV的增殖性表达中， 可转录生成 6种 mRNA， 分别由启动子 P5、 P19、 P40启动合成 （如图 7) 。 在腺病毒 （Ad) 共转染的情况下， Ad的 E1A基因负 责 AAV基因表达的反式激活； Ad E2A基因编码单链 DNA结合蛋白， 可刺激 AAV启动子所启动的转录， 还可促进 AAV转录后从胞核向胞质转运； 另外 Ad VA1 R A可能也有利于 AAV蛋白合成的起始。而 AAV也能够正调节和负调节 自身基因和辅助病毒基因的表达。 AAV的 rep基因能够正调节和负调节 P5、P19、 P40启动子所启动的转录， 在有 Ad的情况下， rep基因产物行使正调节功能， 这 是 AAV基因大量合成所必须的； 而在无辅助病毒共转染的情况下， rep基因产 物则起负调节作用。
在无辅助病毒的情况下， AAV DNA以双链形式整合到宿主细胞基因组中， 在那里以潜伏状态持续存在，形成 AAV的潜伏感染。 AAV整合的特异位点是在 人染色体的 19ql3.3— 19q ter上。 Rep基因产物除了负调节作用外， 还可识别和 结合特异的整合位点， 即 GGTC序列， 并介导了 ITR与整合位点的重组。 而腺 病毒的感染可使其进入增殖感染状态。
2、 AAV作为基因治疗的载体。
AAV是目前已知的唯一能够在宿主基因组特异染色体位点整合的真核细胞 病毒。 这个发现使基因治疗产生了新的希望， 与非整合载体 (如腺病毒为基础的 载体)相比， AAV载体不仅能够更持续稳定地表达转基因， 而且能够减少理论上 由于转基因的随机整合导致的插入突变几率。我们使用的这一套载体系统是将天 然腺相关病毒和腺病毒用分子生物学方法经一系列剪切和修饰而成，它们保留了 腺相关病毒复制和转录所必需的部分， 也保留了腺病毒的晚期基因辅助成份，其 他多余的部分均已删除， 既能实现病毒长期稳定的潜伏感染， 又能避免腺病毒的 致病危险，而且腺病毒晚期基因的表达可以保证被克隆基因的表达效率。这一载 体系统的构成如下-
, pXX2: (如图 8 )保留了 AAV的 cap基因和 rep基因（此时得到 pACG-2 )， 并在下游区域又插入了一个 p5启动子（在原来区域用 Xbal和 Pstl切下， 再连接 到 pACG-2的适当位置） 。
PXX6: (如图 8 ) 在腺病毒改造质粒 ρΒΗΙΟ (已切掉了 El、 E3基因和包装 信号)上用 Pmel和 Sgfl切下一个 8kb的片段（得到 pXX5)，而 pXX6则是由 pXX5 上用 Clal和 Sail切下一段克隆入 pBS中得到的， 仅仅留下 E2A、 E4和 VA编码 区。
pXXUF, (如图 9)： rAAV包装所用的质粒 pXXUF,含有 CMV启动子 (箭头） 所驱动的人 gfp基因， 它是将腺相关病毒的编码基因全部删除后再由 Notl位点 导入的。 为了便于基因克隆和表达， 又将其加工成环状。
二、 rAAV-anti2J2重组病毒的包装与回收
pXX2 、 pXX6、 pXXUFi-an^JS质粒的大量提取， 所用大提试剂盒购自 Promega公司。 方法依说明书所述。
将 293细胞 （人肾母胚胎肿瘤细胞系） 传入数个 150mm培养板中， 传代培 养需要 0.25%胰蛋白酶、高糖 DMEM培养基、新生小牛血清（购自 Gibco公司）。 待细胞生长到 60%到 70%聚集度时， 按 85 g DNA/150mm培养盘的总量， 按摩 尔比 1:1 :1 (质量比为 1.7:3.8:1 )用钙磷转染法转入 293细胞。首先分别将 pXX2、 pXX6、 pXXUFi-antiS 质粒加入一无菌的 250ml培养瓶中， 然后依次加入 2.5MCaCl2溶液（160μ1 /盘）和无菌去离子水，最后加入 2 X BBS (pH6.95， 1.6ml/ 盘） ， 逐滴加入振摇混合液， 以形成较小的钙磷颗粒。 之后室温下孵育 30 ίη。 随即吸弃 293细胞培养盘中的培养基， 加入 3ml DNA—钙磷混合液， 于 35°C、 3%C02培养 16~24小时后换液， 转入 37°C， 5%C02继续培养 36~48小时。 转 染后 48-72小时吸弃所有培养基，加入少量 PBS缓冲液，刮取盘中 293细胞， -80
°C冻存。
为祛除细胞中的异型蛋白， 须用肝素柱纯化， 方法如下： 将收集的含病毒的 293细胞反复冻溶三次， 加入 O.lmg的 DNase I及 O.lmg的 RNase A， 37°C抚育 2小时； 再加入 0.5%的脱氧胆酸， 37°C抚育 30分钟； 离心数分钟， 上清分别经 5μΐΏ和 0.8μηι的滤膜过滤； 取 8ml肝素琼脂糖混悬液加入一直径为 2.5cm装有 阀门的玻璃柱中； 待琼脂糖混悬液流尽后， 在形成的琼脂床上放置一滤膜， 用 25mlPBS(PH 7.4)平衡琼脂床； 关闭阀门， 将上述经过过滤的病毒原液加入玻璃 柱中， 打开阀门，控制病毒以 1滴 /秒的速度滴下； 待病毒液滴完后， 用 25mlPBS (PH7.4) +0.1M NaCl洗两次； 用 15mlPBS (PH7.4 ) +0.4M NaCl洗脱病毒； 用 Millipore Biomax-lOOk NMWL过滤装置 （UFV2BHK40) 浓缩病毒液至 3~5ml。
实施例 3 rAAV-anti2J2病毒滴度的测定一斑点杂交
(1) CYP2J2 cDNA探针的标记和纯化：
同位素标记的核苷酸 α -P32 购自北京雅辉公司。 用 Not I 切下 PXXUF,-anti2J2中的 CYP2J2片段做为已知序列的探针。随机引物法标记 CYP2J2 片段并纯化 （试剂盒购自 QIAGEN公司， 方法如说明书）。
(2)回收物的和定量标准的准备- 回收物经适量 DNase I和 RNase A消化细胞基因组的 DNA和 R A后再加 Proteinase K， 消化病毒的蛋白质外壳 （以上试剂均购自日本 TAKARA公司 )。 酚： 氯仿： 异戊醇 （25:24:1 ) 抽提， 祛除沉淀， 上清中为病毒中的 DNA。 将上 清按体积比对倍稀释。 标准物采用目的基因片段， 即 CYP2J2片段， 测定浓度后 按分子数对倍稀释 （具体计算方法略）。 并作加热和碱变性处理。
(3)杂交与放射自显影
配好斑点杂交装置， 并按装置大小安置一块 0.45 μ ηι的尼龙膜，将待测病毒 样品和标准样品按体积梯度和分子梯度分别上样， 上样后将膜 8CTC干烤 2小时， 后 42°C预杂交 1小时 （预杂交液购自 INTERGEN公司）。 再加加热处理的标记 探针， 45°C杂交 12-16小时。 洗膜后 -80°C放射自显影 2-3天。 制备的 rAAV滴度 达 1 X lOUp.f.u. , 即可用于动物实验。
实施例 4 CYP表氧化酶对肿瘤生长及侵蚀的影响
( 1 ) CYP表氧化酶基因转染后在皮下移植瘤和在肿瘤细胞系中的有效表达 将 control组和转染 rAAV-GFP、 rAAV-anti2J2、 rAAV-CYP2J2组裸鼠皮下移 植瘤和肿瘤细胞系组织蛋白提取后采用 western blot法检测组织中表氧化酶的表 达。 结果显示转染 rAAV-anti2J2组与对照组比较表达明显减少， 转染 rAAV-GFP 组与对照组比较没有显著差异。 见图 10 和8。
(2 ) CYP表氧化酶及抑制剂对四种肿瘤细胞体外培养增殖的影响 将 rAAV-CYP2J2和 rAAV-CYPF87V转染肿瘤细胞，使其高效表达代谢花生 四烯酸产生内源性 EETs的表氧化酶， 间接增加细胞内 EETs浓度， 可直接证实 CYP表氧化酶促增殖作用。与对照组比较，转染 rAAV-CYP2J2和 rAAV-CYPF87V 能明显促进肿瘤细胞的增殖，细胞增殖数是基础状态即转染 rAAV-GFP和未转染 细胞的 2〜3倍（PO.01 ); 转染反义 CYP表氧化酶基因 （rAAV-anti2J2)和加用 CYP表氧化酶抑制剂 17-ODYA后， 细胞增殖明显受抑， 为基础状态下的 50%以 下 (尸 <0.01 )。 见图 11。
(3 ) rAAV-antiCYP表氧化酶对人舌鳞癌细胞裸鼠移植瘤生长的影响 我们采用离体途径将 rAAV-antiCYP转染人舌鳞癌细胞株 Tca-8113， 然后移 植到裸鼠皮下， 部分模拟肿瘤细胞在体内的生长过程， 皮下接种后第 7d、 12d、 17d、 22d、 27d、 32d用游标卡尺精确测量瘤体最长径 (a)与最短径 (b)， 肿瘤体积 V= 7i ab2/6， 绘制肿瘤体积生长曲线。结果如下： 裸鼠接种 Tca-8113后， 经过 4~7 天的潜伏期即出现肉眼可见的皮下瘤结节， 之后， 瘤体生长逐渐加速。各组移植 瘤生长曲线呈 S形， 生长高峰在 12— 22天之间不等。 随着时间的延长， 各组形 成皮下移植瘤的体积出现明显差异。转染 rAAV-antiCYP2J2组出现肿瘤生长延迟 现象， 生长曲线呈 S形右移。 而在转染 rAAV-anti2J2第 32天， 肿瘤的体积的大 小是基础状态的 2倍左右。 这说明 CYP表氧化酶能明显促进在体肿瘤的生长， 抑制其基因表达后能明显抑制肿瘤的生长。 结果如图 12:
(4) . rAAV-AS-CYP表氧化酶对肿瘤组织微血管密度的影响
接种 32d 后处死动物， 剥离瘤体制备石蜡切片行微血管密度 (microvessel density, MVD)计数分析。 显微镜下可见微血管主要分布在瘤组织的间质和周边 区域，而在肿瘤组织内很少有微血管分布。转染 rAAV-CYP2J2和 rAAV-CYPF87V 组微血管增多， 血管密度较大， 在高倍视野下的 MVD分别为 76.8±9.1、 70.2 ±7.8， 与对照组（42.8±6.4)比较有显著性差异（P<0.01 )，而转染 rAAV-anti2J2 组 （23.6± 7. 3 ) 与对照组和转染 i'AAV-CYP2J2 组比较， 微血管密度显著降低 (尸<0.05和尸 <0.01 )。 见图 13。
(5) . rAAV-anti2J2表氧化酶对肿瘤细胞迁移浸润生长的影响
我们釆用了目前常用的测定上皮细胞迁移运动的方法， 即在改良的 Boyden 趋化小室中进行。 μπι 微孔多聚碳酸酯滤膜预先经 IV 型胶原 （collagen IV, lO g/ml) 和 Matrigel(l Wfilter)于 4口中包被 24h。 Tca-8113, A549、 Ncl-H446 和 HepG2细胞系用 DMEM培养液洗涤后调整为 2xl06/ml。在趋化小室的下层加 入 200μ1含 5%FBS的 DMEM培养基作为趋化因子， 然后盖上多聚碳酸酯滤膜， 在趋化小室的上层加入细胞悬液 800μ1， 5% C02， 37口培养 5h后拆卸装置， 收 取滤膜。 擦净滤膜上层面的细胞， 将膜用甲醇固定， 苏木素染色。 在 200倍显微 镜下随机观察 5个视野 /张膜， 计数迁移细胞数。 经多次重复实验结果证实， 转 染 rAAV-antiCYP2J2和加用 CYP表氧化酶抑制剂 17-ODYA后，细胞迁移侵袭能 力明显下降， 约为基础状态下的 60% ( <0.01)。 见图 14A、 B。
此结果说明 rAAV-anti2J2病毒及 CYP表氧化酶抑制剂 （17-ODYA) 具有抑 制肿瘤生增殖转移的作用， 可用于肿瘤治疗。 表达人类 CYP2J2反义基因的重组 腺相关病毒 （rAAV-anti2J2) 的构建成功， 在生物体内可长期高效表达， 从基因 水平上阻断 CYP2J2基因的转录与翻译， 使 CYP2J2表达减少， 其作用更强， 更 为彻底。 将其与相信表达人类 CYP2J2 反义基因的重组腺相关病毒 (rAAV-anti2J2 )和 CYP表氧化酶抑制剂用于临床后，必将在肿瘤等严重危害人 类健康的疾病的治疗中， 发挥极其主要的作用。
实施例 5 rAAV- anti2J2表氧化酶对肿瘤细胞凋亡的影响
按照上述方法分别用 l-AAV-GFP, rAAV-anti2J2, rAAV-F87V和 rAAV-CYP2J2 转染四种不同的肿瘤细胞系， 一周后用 TNFa诱导细胞凋亡， 然后分别用不同的 方法检测细胞凋亡情况。 通过 DNA ladder^ 丫啶橙染色和流式细胞术方法分析 发现： ①直接加入 EETs或转染 rAAV-CYP2J2， rAAV-CYPF87V均能明显减少 DNA ladder形成， 而使用表氧化酶抑制剂 17-ODYA或转染反义 CYP2J2则明显 促进 诱导的肿瘤细胞凋亡； ②细胞用丫啶橙 /溴化乙啶染色后， 在荧光 显微镜下可见转染表氧化酶基因和直接用 EETs干预的细胞脱落少，细胞密度高， 细胞凋亡数明显较对照组少，而用 17-0DYA和转染反义表氧化酶基因的肿瘤细胞 脱落明显， 密度明显降低， 大多细胞核染色质浓缩、 断裂呈颗粒状， 细胞凋亡明 显增多。 这从形态学改变初歩说明 EETs和表氧化酶基因具有抗 TNF-oi诱导肿瘤 细胞凋亡的作用；③分别用 PI单染法和 Annexin V-FITC/PI双染色后通过流式细 胞仪分析 TNF-α诱导的细胞总凋亡率和早期凋亡率。 结果显示： 用 3种 EETs预 处理过的肿瘤细胞总凋亡率分别为 14.94% ±3.5、 11.72%±4.2、 12.89%±2.7， 显著低于对照组 （28.15%±4.7) 和溶媒组 （27.86%±6.1， P均 <0.01 )， 更低于 17-ODYA组 （52.65%±3.6)； 转染 CYP2J2和 CYPF87V的肿瘤细胞总凋亡率分 别为 7.24%±4.2和 7.79%±2.7， 明显较对照组的 20.15%±3.5和 17-ODYA组的 33.51%±6.1及转染反义 CYP表氧化酶基因组的 40.74%士 3.5低 (P<0.01 ) ; ④ 对表氧化酶和 EETs抗凋亡机制的研究显示： EETs和转染表氧化酶基因均能非常 显著地增加 Akt磷酸化， 上调细胞抗凋亡蛋白 Bcl-2、 Bcl-xL的表达， 同时下调 凋亡蛋白 Bax的表达； 而使用 CYP表氧化酶抑制剂或转染 rAVV-anti2;T2则产生 完全相反的作用。 这些结果表明， 表氧化酶和 EETs不仅显著促进肿瘤生长， 而 且还可通过抑制 TNF-CC诱导的细胞凋亡来保护肿瘤细胞。因此根据这些结果，可 以使用 CYP表氧化酶抑制剂或转染 rAVV-anti2J2促进肿瘤凋亡从而达到治疗肿 瘤的目的。

Claims

1、一种表达人类 CYP2J2反义基因的重组腺相关病毒，其中该重组腺相关病 毒包括的三种质粒：
1 ) pX¾: 为包装质粒， 含有编码腺相关病毒 Rep蛋白和 Cap蛋白的编码序 列。 并在上游和下游各插入一个 p5启动子， 使表达效率可以提高 15倍， 提供 rAAV复制所必须.的 Rep蛋白；
2) pXXUF,: 真核表达载体， 含 CMV启动子， 有较强的表达效率， 其含有一 多克隆 Not I位点， 可以连接不同的目的基因， pXXUF,反向连接人 CYP表氧化酶 权
基因， 该载体含有 rAAV转基因表达所必须的末端重复序列（ITRs), 负责病毒的 复制和病毒外壳的包被并携带目的基因；
3 ) pXXa: 为辅助质粒， 删除了腺病毒的致病基因序列， 保留了腺病毒 E1A、 E2A和 VA, RNA基因，'所表达的蛋白质可发挥辅助作用以刺激 rAAV基因 β¾转录和 翻译， 保证 rAAV产量；
4 ) 以上三种质粒经磷酸钙沉淀法共转染 293求细胞， 即可在 293细胞内形成 高滴度的含目的基因的 rAAV颗粒。
2、 一种表达人类 CYP2J2反义基因的重组腺相关病毒的制备， 其步骤：
1 ) X 质粒——提供腺相关病毒包装所必需的 Rep蛋白和 Cap蛋白的编码序 列， 和 pXXe质粒——提供刺激腺相关病毒复制和转录所必需的腺病毒 E1A、 E2A 和 VAI RNA基因；
2 ) 含腺相关病毒基因组的真核表达载体 pXXUF, , 在该真核载体中腺相关病 毒缺失 cap蛋白和 rep蛋白编码区， 并插入人类 CYP2J2反义基因， 形成重组质 粒 pXXUF,- anti2J2;
3 )步骤 1 ) 中的 pXX2、 pX 和步骤 2 )中的 pXXUF,- anti2J2通过磷酸钙沉淀 法共转染 293细胞， 适时回收， 纯化、 斑点杂交法检测其病毒滴度；
4) 歩骤 3) 中回收纯化的病毒转染肿瘤细胞， 检测其表达及生物学活性。
3、权利要求 1所述的表达人类 CYP2J2反义基因的重组腺相关病毒， 该重组 腺相关病毒由天然状态的腺相关病毒经分子生物学方法人工剪切、 修饰和加工， 再经分子生物学方法包装复制， 并经纯化而成。
4、 一种药物组合物， 其特征在于， 它包括有效量的权利要求 1所述的表达 人类 CYP2J2反义基因的重组腺相关病毒和药学上所接受的载体或赋形剂。
5、 如权利要求 1所述的一种表达人类 CYP2J2反义基因的重组腺相关病毒， 通选择性阻断肿瘤组织中 CYP2J2基因表达， 抑制表氧化二十垸三烯酸合成而治 疗恶性肿瘤。
6、如权利要求 1、 5所述一种表达人类 CYP2J2反义基因的重组腺相关病毒， 选用 CYP表氧化酶选择性抑制剂和 CYP表氧化酶代谢花生四烯酸所形成的表氧化 产生表氧化二十垸三烯酸的拮抗剂，这些及类似的化学制剂是人工化合而成并具 有选择性抑制 CYP表氧化酶系活性或阻断表氧化二十烷三烯酸的作用，能够控制 肿瘤增殖， 而对细胞并无毒性作用。
7、 如权利要求 1所述的一种表达人类 CYP212反义基因的重组腺相关病毒， 已保藏于武汉大学内中国典型物保护中心， 保藏日期： 2004年 6月 30日， 保藏 编号： CCTCCNO: V200411分类命名： 表达人类细胞色素 P4502J2反义基因的重 组腺相关病毒。
PCT/CN2005/002040 2004-11-30 2005-11-28 Virus adéno-associé recombinant exprimant le gène antisens humain cyp2j2 et méthodes de préparation dudit virus WO2006058489A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US11/720,035 US8273344B2 (en) 2004-11-30 2005-11-28 Recombinant adeno-associated virus expressing human antisense gene CyP2J2 and its preparation methods

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN200410061237.7 2004-11-30
CNB2004100612377A CN1286981C (zh) 2004-11-30 2004-11-30 表达人类cyp2j2反义基因的重组腺相关病毒及其制备方法

Publications (1)

Publication Number Publication Date
WO2006058489A1 true WO2006058489A1 (fr) 2006-06-08

Family

ID=35007340

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2005/002040 WO2006058489A1 (fr) 2004-11-30 2005-11-28 Virus adéno-associé recombinant exprimant le gène antisens humain cyp2j2 et méthodes de préparation dudit virus

Country Status (3)

Country Link
US (1) US8273344B2 (zh)
CN (1) CN1286981C (zh)
WO (1) WO2006058489A1 (zh)

Families Citing this family (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB201210357D0 (en) 2012-06-12 2012-07-25 Ucl Business Plc Factor VIII sequences
US9504762B2 (en) * 2013-09-12 2016-11-29 Biomarin Pharmaceutical Inc. Adeno-associated virus factor VIII vectors
WO2015191508A1 (en) 2014-06-09 2015-12-17 Voyager Therapeutics, Inc. Chimeric capsids
JP6401871B2 (ja) 2014-11-05 2018-10-10 ボイジャー セラピューティクス インコーポレイテッドＶｏｙａｇｅｒ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． パーキンソン病の治療のためのａａｄｃポリヌクレオチド
KR20230169197A (ko) 2014-11-14 2023-12-15 보이저 테라퓨틱스, 인크. 근위축성 측삭 경화증(als)을 치료하는 조성물 및 방법
KR102584655B1 (ko) 2014-11-14 2023-10-06 보이저 테라퓨틱스, 인크. 조절성 폴리뉴클레오티드
US11697825B2 (en) 2014-12-12 2023-07-11 Voyager Therapeutics, Inc. Compositions and methods for the production of scAAV
WO2017096162A1 (en) 2015-12-02 2017-06-08 Voyager Therapeutics, Inc. Assays for the detection of aav neutralizing antibodies
WO2017189959A1 (en) 2016-04-29 2017-11-02 Voyager Therapeutics, Inc. Compositions for the treatment of disease
WO2017189964A2 (en) 2016-04-29 2017-11-02 Voyager Therapeutics, Inc. Compositions for the treatment of disease
SG11201809699XA (en) 2016-05-18 2018-12-28 Voyager Therapeutics Inc Modulatory polynucleotides
WO2017201258A1 (en) 2016-05-18 2017-11-23 Voyager Therapeutics, Inc. Compositions and methods of treating huntington's disease
CN110650673B (zh) 2016-08-30 2024-04-09 加利福尼亚大学董事会 用于生物医学靶向和递送的方法以及用于实践该方法的装置和系统
EP3619308A4 (en) 2017-05-05 2021-01-27 Voyager Therapeutics, Inc. COMPOSITIONS AND METHODS OF TREATMENT FOR HUNTINGTON'S MORBUS
AU2018261790A1 (en) 2017-05-05 2019-11-28 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)
JOP20190269A1 (ar) 2017-06-15 2019-11-20 Voyager Therapeutics Inc بولي نوكليوتيدات aadc لعلاج مرض باركنسون
CN116891534A (zh) 2017-06-27 2023-10-17 瑞泽恩制药公司 向性修饰的重组病毒粒子和其用于将遗传物质靶向引入至人类细胞中的用途
CN107475291A (zh) * 2017-07-03 2017-12-15 华东师范大学 一种高效表达人源cyp2j2重组酶的方法和应用
CN111132626B (zh) 2017-07-17 2024-01-30 沃雅戈治疗公司 轨迹阵列引导系统
TWI832036B (zh) 2017-08-03 2024-02-11 美商航海家醫療公司 用於aav之遞送之組合物及方法
US11434502B2 (en) 2017-10-16 2022-09-06 Voyager Therapeutics, Inc. Treatment of amyotrophic lateral sclerosis (ALS)
US20200237799A1 (en) 2017-10-16 2020-07-30 Voyager Therapeutics, Inc. Treatment of amyotrophic lateral sclerosis (als)
CN108531496B (zh) * 2018-04-04 2020-11-06 江南大学 一种提高外源基因mRNA数量的DNA及其应用
CN114657153A (zh) * 2022-05-24 2022-06-24 上海健士拜生物科技有限公司 rAAV重组包装质粒、用于rAAV包装的质粒系统和rAAV的制备方法
CN116376843B (zh) * 2023-05-24 2023-08-18 济南宜明医疗科技有限公司 腺相关病毒易感性细胞株及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997012050A1 (en) * 1995-09-27 1997-04-03 The Rockefeller University Method for transferring genes to the heart using aav vectors
CN1233291A (zh) * 1996-09-06 1999-10-27 宾西法尼亚大学托管会 重组腺伴随病毒定向基因治疗的方法
CN1422950A (zh) * 2002-04-28 2003-06-11 华中科技大学同济医学院附属同济医院 表达人类血管紧张素ii受体i反义基因的重组腺相关病毒及其制备方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030092658A1 (en) * 2001-02-02 2003-05-15 Meyers Rachel E. Novel human enzyme family members and uses thereof
KR100436089B1 (ko) * 2000-07-06 2004-06-14 설대우 분비성 재조합 트라이머형의 트레일 단백질 생산을 위한 디엔에이 카세트, 테트라사이클린/독시사이클린-유도성아데노-관련 바이러스 벡터, 이 둘의 조합, 및 이들을이용한 유전자 치료
DE50213170D1 (de) * 2002-09-04 2009-02-12 Fraunhofer Ges Forschung Verwendung eines Mittel zur Therapie der Herzhypertrophie

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997012050A1 (en) * 1995-09-27 1997-04-03 The Rockefeller University Method for transferring genes to the heart using aav vectors
CN1233291A (zh) * 1996-09-06 1999-10-27 宾西法尼亚大学托管会 重组腺伴随病毒定向基因治疗的方法
CN1422950A (zh) * 2002-04-28 2003-06-11 华中科技大学同济医学院附属同济医院 表达人类血管紧张素ii受体i反义基因的重组腺相关病毒及其制备方法

Also Published As

Publication number Publication date
CN1657632A (zh) 2005-08-24
US8273344B2 (en) 2012-09-25
CN1286981C (zh) 2006-11-29
US20080131403A1 (en) 2008-06-05

Similar Documents

Publication Publication Date Title
WO2006058489A1 (fr) Virus adéno-associé recombinant exprimant le gène antisens humain cyp2j2 et méthodes de préparation dudit virus
US11618888B2 (en) Tumor-selective E1A and E1B mutants
Bingle et al. Cytokine-Mediated Induction of the Human Elafin Gene in Pulmonary Epithelial Cells Is Regulated by Nuclear Factor-κ B
JPH10503361A (ja) 組換えｐ５３アデノウイルス方法と組成物
CN110484615B (zh) lncRNA在病毒性心肌炎中调控巨噬细胞极化的应用
Zhang et al. Long non‐coding RNA AK085865 ablation confers susceptibility to viral myocarditis by regulating macrophage polarization
WO2012161352A1 (ja) Reic発現アデノウイルスベクター
EP1121441A2 (en) Selectively replicating viral vectors
AU2007243252A1 (en) An isolated DNA fragment of the Sparc human promoter and its use
CN110885798B (zh) 靶向前列腺癌的重组溶瘤腺病毒及其构建方法和应用
JP2003504316A (ja) 疾患を処置するためのアデノウイルスベクター
CN117568347B (zh) Ppef1作为神经母细胞瘤药物靶点的应用
CN109908368A (zh) 一种senp1基因和/或senp1蛋白在制备治疗心血管疾病药物中的应用
CN112126631A (zh) 携带ERCC1基因siRNA的双调控肿瘤特异性溶瘤腺病毒
Wang et al. Construction of Repulsive Guidance Molecule A-Specific RNA Interference Recombinant Adenovirus
WO2012121071A1 (ja) VA-RNAsが発現しないアデノウイルスベクター
CN116904517A (zh) 增强肝细胞增殖能力或培养肝细胞的方法及其应用
CN115029789A (zh) DC-SIGN的shRNA库的构建及其在原发性肝癌中的应用
CN111118139A (zh) 一种骨质疏松的分子靶标及其应用
Abbas UNDERSTANDING THE RELATIONSHIP BETWEEN ONCOLYTIC AD5 DELETED E1b55KDA LYTIC INFECTION AND P53 IN MAMMALIAN CELLS
CA2277615A1 (en) Sv40 viral vectors for targeted integration into cells
KR19980074610A (ko) 고순도의 활성화된 미토겐 활성 단백질 키네이즈 키네이즈-1의 제조 방법

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 11720035

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 05814299

Country of ref document: EP

Kind code of ref document: A1

WWP Wipo information: published in national office

Ref document number: 11720035

Country of ref document: US