[go: up one dir, main page]

WO2001037867A1 - Nouvel adenovirus defectif et son procede d'obtention - Google Patents

Nouvel adenovirus defectif et son procede d'obtention Download PDF

Info

Publication number
WO2001037867A1
WO2001037867A1 PCT/CN2000/000428 CN0000428W WO0137867A1 WO 2001037867 A1 WO2001037867 A1 WO 2001037867A1 CN 0000428 W CN0000428 W CN 0000428W WO 0137867 A1 WO0137867 A1 WO 0137867A1
Authority
WO
WIPO (PCT)
Prior art keywords
adenovirus
deletion
defective
cells
defective adenovirus
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CN2000/000428
Other languages
English (en)
Chinese (zh)
Inventor
Qijun Qian
Jonathan Sham
Mengchao Wu
Xiaoyan Che
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GENE BIO-TECH Corp Ltd
Original Assignee
GENE BIO-TECH Corp Ltd
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 GENE BIO-TECH Corp Ltd filed Critical GENE BIO-TECH Corp Ltd
Priority to AU13794/01A priority Critical patent/AU1379401A/en
Publication of WO2001037867A1 publication Critical patent/WO2001037867A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A61K35/761—Adenovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to the field of life sciences, and is a defective adenovirus vector system for treating tumors and a construction scheme thereof.
  • the object of the present invention is to provide a defective adenovirus with a novel mutation of the E1b 55Kda protein gene and a new method for constructing the same.
  • Gene therapy is a new method for the treatment of malignant tumors that has emerged in recent years.
  • the gene transfection method is divided into two methods: viral method and non-viral method.
  • Viral method usually uses reverse transcription disease.
  • Retroviruses have a high transfection rate in vitro, but the virus titer is low, the transfection rate is low in vivo, and they can only infect cells in the division stage. At the same time, they have the ability to integrate into the chromosome and have the potential to become cancerous. Disadvantages.
  • Adeno-associated virus has the ability to transfect dividing and quiescent cells, and can be persistently expressed. In contrast, non-viral methods, including liposomes and i-invasion methods, have a shorter transfection gene expression time and a lower transfection rate.
  • Adenovirus is currently the most common viral vector for tumor gene therapy.
  • E1a can inhibit HER-2 / neu over-expressing tumors, including down-regulating HER-2 / neu gene expression, reducing the pathogenicity, inhibiting tumor metastasis, and inducing tumor cells Apoptosis and enhance tumor chemotherapy sensitivity and other functions.
  • E1 including E1a and E1b
  • E1b is missing from the commonly used adenovirus vector system, so that the adenovirus lacks the antitumor effect of E1a.
  • the defective adenovirus of the present invention is a partial deletion of the E1b 55Kda protein gene and a stop codon is inserted at the deletion site, and a 2809-3329bp deletion is inserted into the deletion site.
  • a DNA fragment TMTGAGTMCTM is inserted into the DNA fragment, and the DNA fragment contains two stop codons, respectively TM And TGA.
  • the defective adenovirus of the present invention can be combined with a chemotherapeutic drug such as cisplatin, 5-fluorouracil mitomycin C, etc., a biotoxin such as snake venom, a monoclonal antibody such as an anti-liver cancer cell antibody, etc. Antitumor drugs work better.
  • a chemotherapeutic drug such as cisplatin, 5-fluorouracil mitomycin C, etc.
  • a biotoxin such as snake venom
  • a monoclonal antibody such as an anti-liver cancer cell antibody, etc.
  • Antitumor drugs work better.
  • two adenovirus vectors are transfected into cells that will produce recombination, and one E1b 55Kda protein gene in the two adenovirus vectors is used.
  • the 2809-3329bp deletion inserting a stop codon 'at its deletion site, inserts the DNA fragment TMTGAGTMCTAA, which contains two stop codons, T and TGA, respectively.
  • the human adenovirus has six homogeneous subgenus, divided into A, B, C, D, and E F. Their affinity for host cells, tropism, tumorigenicity, and disease history are not the same.
  • Ad5 subtype 5 Ad5 as an example.
  • the adenovirus system is composed of two vectors, one vector provides the left arm portion of the adenovirus, and the other vector provides the right arm. These two vectors have at least 500 nt homologous recombination regions, but the transfected cells produce recombinant adenovirus,
  • the vector system plasmid pXC1 contains the left arm of wild-type Ad5.
  • pBHGIO provides Ad5 right arm lacking E3 region or pBHGE3 provides Ad5 right arm (including E3 region).
  • the primers for the polymerase chain reaction synthesized in the present invention inserted in the position near the two ends of the linker are
  • the invention transfects the defective adenovirus into E1 transformed human embryonic kidney cell line 293 or E1 transformed human embryonic retinal cell line 911, which can be effectively made to have anti-tumor adenopathy.
  • the invention has the following beneficial effects
  • the present invention provides a defective adenovirus with a novel mutation in the E1b 55Kda protein gene, and animal experiments have proven that the adenovirus can be used to treat a variety of tumors.
  • the invention provides a defective adenovirus with a novel mutation in the E1b 55Kda protein gene. Build method. This method can be used to construct a new type of defective adenovirus. The method is easy for the operator to master, and can be used to construct a variety of new type of defective adenovirus, which has established a good foundation for gene therapy diseases.
  • Example 1 Partial deletion of the adenovirus E1b protein gene and construction of a stop codon vector inserted in the deletion region
  • the pXC.1 vector was purchased from Microbix Biosystem Inc. (Toronto) of Canada.
  • pXC.1 contains a type 5 adenovirus sequence bp22-5790. This vector was cut in 3329bp with endonuclease Bgl II and then partially digested with endonuclease Hind III to recover the 9372bp DNA fragment. The 3 'concave end was smoothed using E. coli DNA polymerase I Klenow fragment, which is The 2809bp-3329bp region was deleted from the pXC.1 vector (see the Molecular Cloning Experiment Guide for specific methods, published by Scientific Publishing 1992).
  • the two DNA oligonucleotide fragments were each mixed with 0.1 g, and denatured at 10 CTC for 5 minutes, and then renatured with hypothermia, and then phosphorylated with T4 phage polynucleotide acid.
  • the acidified adapter was ligated with the pXC.1 vector fragment lacking the 2809-3329bp region and named pXC-del E1b (for the method, see the Molecular Cloning Experiment Guide, Science (Published in 1992).
  • the primers were synthesized in the positions near the two ends of the inserted connector, respectively
  • the defective virus of the present invention is transfected into E1 transformed human embryonic kidney cell line 293 or E1 transformed human embryonic retinal cell line 911 cells. In the present invention, it was transfected into 293 cells.
  • the 293 cell line was purchased from Microbix Biosystem Inc., Canada. (Toronto) is a type 5 adenovirus DNA transformed into human embryonic kidney cells. It contains and expresses the type 5 adenovirus E1 region, which has a high transfection rate.
  • a plasmid containing adenovirus type 5 left arm was used to co-transfect 293 cells with a plasmid containing adenovirus type 5 right arm. Homologous recombination can produce infectious adenoviruses.
  • PBHG10 and pBHG bow 3 purchased from Microbix Biosystems, Canada
  • pBHGIO contains the right arm of type 5 adenovirus, but lacks the E3 region.
  • PBHGE3 contains the right arm of type 5 adenovirus and contains the E3 region.
  • Viral plaques appeared 9-14 days after co-transfection. After three viral plaque purifications, the adenoviruses were named CNHK201 and CNHK202. For specific methods, refer to Gene Transfer and Expression Protocols, edited by Murray EJ, published by Humana Press 1991.
  • Ad5 left arm plasmid Ad5 right arm plasmid
  • Ad5-del E1b CNHK202 pXC-de! E1b PBHGE3 Adenoviruses multiply in 293 cells. Adenoviruses are purified by mass centrifugation using a gaseous cesium gradient centrifugation method. ). Its insertion sequence was confirmed by sequencing.
  • Ad5-del E1b E3 (CNHK201) is a type 5 adenovirus with deletion mutations in the 2809-3329bp region (partial sequence of the E1b 55Kda protein gene), and inserted a sequence containing two stop codons in the deletion mutation region TAATGAGTAACTAA, accompanied by 28133-30818bp (partial sequence of E3 region), other DNA sequences of the virus are the same as type 5 adenovirus.
  • Ad5-del E1b (CNHK202) is a type 5 adenovirus with deletion mutations in the 2809-3329bp region (partial sequence of the E1b 55Kda protein gene), and a sequence containing two stop codons, TAATGAGTAACTAA, is inserted in the deletion mutation region, and other viral DNA sequences Same as adenovirus type 5.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Mycology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Public Health (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention porte sur un nouvel adénovirus défectif présentant un manque partiel de la séquence codant pour la protéine d'adénovirus naturel E1b de 55kDa, sur son procédé d'obtention, et sur des adénovirus défectifs produits d'assemblage de recombinaison présentant un déficit fonctionnel en protéine E1b de 55kDa du à une mutation suppressive combinée à l'insertion d'un codon terminal, et restreignant la fonction des protéines E1a et E1b de 19kDa et d'autres protéines virales. L'E1a et d'autres protéines virales d'adénovirus sont bénéfiques contre les tumeurs; le système de vecteurs d'adénovirus défectifs peut donc servir à la thérapie des tumeurs.
PCT/CN2000/000428 1999-11-19 2000-11-17 Nouvel adenovirus defectif et son procede d'obtention Ceased WO2001037867A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU13794/01A AU1379401A (en) 1999-11-19 2000-11-17 A new defective adenovirus and process for producing said adenovirus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN99124030A CN1254719A (zh) 1999-11-19 1999-11-19 一种缺陷型腺病毒及其构建方法
CN99124030.8 1999-11-19

Publications (1)

Publication Number Publication Date
WO2001037867A1 true WO2001037867A1 (fr) 2001-05-31

Family

ID=5283127

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2000/000428 Ceased WO2001037867A1 (fr) 1999-11-19 2000-11-17 Nouvel adenovirus defectif et son procede d'obtention

Country Status (3)

Country Link
CN (1) CN1254719A (fr)
AU (1) AU1379401A (fr)
WO (1) WO2001037867A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1388248A (zh) * 2001-05-25 2003-01-01 钱其军 高效表达干扰素的肿瘤细胞内特异性增殖的腺病毒及其构建方法
CN1880457B (zh) * 2001-10-11 2010-05-26 麦克公司 Ad6重组核酸
CN101781636A (zh) * 2009-01-19 2010-07-21 中国人民解放军第二军医大学东方肝胆外科医院 一种含11型腺病毒纤毛蛋白基因的增殖型重组溶瘤腺病毒、其构建方法及其用途

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994028152A1 (fr) * 1993-05-28 1994-12-08 Transgene S.A. Adenovirus defectifs et lignees de complementation correspondantes
WO1996012030A1 (fr) * 1994-10-17 1996-04-25 Rhone-Poulenc Rorer S.A. Adenovirus defectifs comprenant un gene therapeutique et un gene immunoprotecteur

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994028152A1 (fr) * 1993-05-28 1994-12-08 Transgene S.A. Adenovirus defectifs et lignees de complementation correspondantes
WO1996012030A1 (fr) * 1994-10-17 1996-04-25 Rhone-Poulenc Rorer S.A. Adenovirus defectifs comprenant un gene therapeutique et un gene immunoprotecteur

Also Published As

Publication number Publication date
CN1254719A (zh) 2000-05-31
AU1379401A (en) 2001-06-04

Similar Documents

Publication Publication Date Title
AU762940B2 (en) Adenovirus vectors containing cell status-specific response elements and methods of use thereof
AU745560B2 (en) Adenovirus vectors specific for cells expressing alpha-fetoprotein and methods of use thereof
JP3995259B2 (ja) 動物起源のアデノウイルスベクターおよび遺伝子治療におけるそれらの使用
CA2283231C (fr) Vecteurs d'adenovirus contenant des elements heterologues regulateurs de transcription, et leur procede d'utilisation
JP2002514075A (ja) 癌胎児性抗原を発現する細胞に特異的なアデノウイルスベクターおよびその使用方法
US7109029B2 (en) Vector constructs
JP2001503617A (ja) 標的化腫瘍遺伝子治療のためのレセプターを介した遺伝子移送系
US20020106746A1 (en) Anti-inflammatory vectors
WO2005035744A1 (fr) Tumeur ciblant un virus a deux genes, procedes d'hybridation et utilisation
WO2001037867A1 (fr) Nouvel adenovirus defectif et son procede d'obtention
US20050271622A1 (en) Construct of tumor-selective recombinant adenovirus, method for preparing the same and use thereof
WO2006125381A1 (fr) Virus du gene de ciblage tumoral zd55-il-24, son procede de construction et son application
AU2003252891B2 (en) Adenovirus vectors containing cell status-specific response elements and methods of use thereof
CN118853767A (zh) 一种新型溶瘤腺病毒载体及其构建方法和应用
KR20030090918A (ko) 개선된 종양 살상 효과를 갖는 재조합 아데노바이러스 및그를 포함하는 약제학적 조성물
WO2004078987A1 (fr) Produit de recombinaison construit a partir d'un vecteur viral et d'un gene humain suppresseur de tumeur et utilisation dudit produit de recombinaison
EP1905837A1 (fr) Vecteurs d'adénovirus contenant des éléments hétérologues régulateurs de la transcription et procédés les utilisant
EP0968298A2 (fr) Vecteurs d'adenovirus specifiques de cellules exprimant l'antigene carcino-embrionnaire et leurs methodes d'utilisation
AU2002252061A1 (en) Vector constucts
CA2282708A1 (fr) Vecteurs d'adenovirus specifiques de cellules exprimant l'antigene carcino-embrionnaire et leurs methodes d'utilisation

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

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

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase