WO2002038783A1 - Vecteur - Google Patents

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Publication number
WO2002038783A1
WO2002038783A1 PCT/GB2001/004916 GB0104916W WO0238783A1 WO 2002038783 A1 WO2002038783 A1 WO 2002038783A1 GB 0104916 W GB0104916 W GB 0104916W WO 0238783 A1 WO0238783 A1 WO 0238783A1
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WO
WIPO (PCT)
Prior art keywords
component
donor
expression
recombination
vector
Prior art date
Application number
PCT/GB2001/004916
Other languages
English (en)
Inventor
Johann Winkler
Kenneth Robert Bundell
Original Assignee
Astrazeneca Ab
Astrazeneca Uk Limited
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
Priority claimed from GBGB0027501.6A external-priority patent/GB0027501D0/en
Application filed by Astrazeneca Ab, Astrazeneca Uk Limited filed Critical Astrazeneca Ab
Priority to JP2002542098A priority Critical patent/JP2004513638A/ja
Priority to EP01980719A priority patent/EP1337656A1/fr
Priority to AU2002212507A priority patent/AU2002212507A1/en
Priority to US10/416,128 priority patent/US20040048381A1/en
Publication of WO2002038783A1 publication Critical patent/WO2002038783A1/fr

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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
    • 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
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • Adenoviruses are a group of DNA viruses which can cause generally mild infections in humans respiratory illness, conjunctivitis and infantile gastroenteritis. Almost grown in cell culture, adenoviruses have been widely studied for many years (e.g. RNA splicing was first described in adenovirus infected cell). The ability of adenoviruses to infect human cells at up to 100% efficiency has led to its use as a vector for introducing foreign (recombinant) DNA in both cell culture and in humans (gene therapy). Recombinant adenoviruses generally have certain regions of DNA deleted (e.g. El region necessary for replication, E3 required for evading host immunity). The purpose of this is twofold.
  • the removal of non-necessary regions of DNA allows space for the introduction of foreign DNA which can then be packaged as adenovirus DNA and subsequently introduced into human cells (there is also some leeway for insertion of extra DNA into the genome without affecting infectivity).
  • the removal of the El region determines that the recombinant virus can infect human cells but not replicate; this is an important safety consideration.
  • DNA is transfected into a cell line (e.g. HEK 293) which has the El region engineered into its genome i.e. the cell line provides the replicative machinery lacking in the recombinant adenovirus.
  • Recombinant adenovirus technology is often based around human adenovirus type 5.
  • the genome for Ad5 is 35.935 kb. This whole sequence can be cloned into a plasmid vector and replicated in E.coli. However, because of the length of adenovirus sequence, there are very few suitable restriction enzyme sites available which would allow direct cloning into such a vector. To circumvent this problem, a two vector approach has been adopted. One vector contains the complete adenovirus sequence minus El and E3 sequence. The second vector contains a eukaryotic promoter (e.g.
  • CMN CMN upstream of a cloning site (for insertion of foreign D ⁇ A), and polyadenylation sequences necessary for stability of transcribed R ⁇ A. Flanking this expression cassette are two regions of adenovirus D ⁇ A which are common with adenovirus sequences in the first vector.
  • the expression cassette from the second vector can be introduced into the adenovirus sequence in the first vector by a process of homologous recombination. This tedious, exacting and time-consuming process has traditionally been performed in HEK 293 cells. Viruses produced have to be plaque purified, propagated and tested to ensure that the desired recombinant D ⁇ A has been introduced.
  • Recombinant adenoviral DNA can then be identified by restriction digest and clonal adenoviral DNA prepared in E.coli.
  • the clonal adenoviral DNA can then be transfected into HEK 293 cells and adenovirus produced thus effectively removing the need for plaque purification.
  • This method has now been commercialised (Q-biogene: AdeasyTM).
  • AdeasyTM Q-biogene
  • a further improvement with this system is that the recombinant adenovirus co-expresses green fluorescent protein from a second CMV promoter. This makes infection efficiency (and therefore gene delivery efficiency) simple to assess.
  • Our experience with the AdeasyTM system has shown that there can be difficulties with the recombination procedure.
  • a two component system for in vitro cloning of a heterologous polynucleotide into adenoviral DNA comprising: i) a first component which is an insert donor comprising a heterologous polynucleotide encoding a heterologous polypeptide; and ii) a second component which is a vector donor comprising an adenovirus genome and an expression cassette; wherein the insert donor and vector donor are adapted for site specific recombination for insertion of the heterologous polynucleotide into the expression cassette capable of forming an adenoviral expression clone in vitro in the presence of a suitable recombination mediator protein or proteins.
  • the site specific recombination uses recombination sites from phage lambda.
  • recombination sites from phage lambda.
  • the reader is referred to the following: Landy (1989) Ann Rev Biochem 58, 913; and Ptashne (1992) A Genetic Switch, Cell Press, Cambridge.
  • the reader is also referrred to US 5888732 (Life Technologies) for further technical details of site specific recombination using insert donor and vector donor moieties.
  • the recombination reactions produce highly specific cutting and ligation reactions such that the recombination mediator proteins cut to the left and right of the heterologous polynucleotide in the insert donor and ligate it into the vector donor whereby to form an adenoviral expression clone.
  • the expression cassette comprises a polynucleotide encoding a fluorescent protein downstream of an internal ribosome entry site for expression from the same mRNA as the heterologous polypeptide.
  • system described herein comprises at least one of the following:
  • a vector donor comprising a ccdB gene
  • an insert donor comprising a selectable marker
  • the system comprises all of the elements i) to vi).
  • the attB x attP reaction is mediated by proteins it and HF (Clonase BPTM, Life Technologies).
  • the attL x attR reaction is mediated by proteins L t, IHF, and Xis (Clonase LRTM, Life Technologies). Lit and Xis are from lambda; IHF is from E. coli.
  • "x" represents recombination.
  • Engineered recombination sites offereing efficiency or specificity advantages over wild type sequences as described in US 5888732 are also contemplated.
  • the method uses first and second components as defined in elements i) to vi) above, the host organism for expression clone replication is E. coli and the host organism for adenoviral replication is HEK 293 cells.
  • Figure 3 shows in vitro cloning of lac Z into an adenoviral expression clone
  • E Lit, IHF, Xis proteins.
  • F Will not grow on ampicillin or in E.coli DH5 ⁇ (ccdB lethal).
  • G Transform E.coli (eg. DH5 ⁇ ) and select on ampicillin plates identify correct clones and transfect HEK 293 cells to generate virus.
  • a vector which contains: the complete adenovirus genome (minus El and E3); an expression cassette comprising: CMN promoter, attRl - Chloramphenicol resistance -ccdB -attR2, internal ribosome entry site (IRES), fluorescent protein sequence, and SN40 polyadenylation sequence.
  • This vector is a vector donor. Any sequence in an insert donor can be efficiently cloned directly into this vector donor via in vitro recombination. Background is reduced to zero due to the ccdB gene toxicity in E.coli (the vector donor D ⁇ A is propagated in E.coli strain DB3.1 which has a gyrA mutation and tolerates ccdB). Thus in vitro recombination of the gene of interest in an insert donor into the vector donor followed by transformation in E.coli DH5a using selection for the vector donor (vector donor ampicillin resistant, insert donor kanamycin resistant) should only result in recombinant adenoviral D ⁇ A since the ccdB gene is toxic.
  • the D ⁇ A is then digested to remove the plasmid backbone and directly transfected into HEK 293 cells to generate recombinant virus.
  • This system to generate recombinant expressing lacZ from an insert donor containing lacZ. It is quick and efficient and we believe has a considerable advantage over the current AdEasy system.
  • these constructs express a fluorescent protein from an IRES element. This means that they are expressed from the same transcribed messenger R ⁇ A as the recombinant gene. With 'AdEasyTM', the green fluorescent protein is expressed from a second separate CMN promoter. Li this case expression of the fluorescent protein is no guarantee of recombinant gene transcription.
  • a 3168 base pair fragment comprising the entire coding region of the E.coli lacZ gene preceded by a sequence encoding six histidine residues was isolated from pZeoSN2/lacZ (Livitrogen) by restriction enzyme digest ( ⁇ co I and EcoR I).
  • the lacZ D ⁇ A fragment was separated from the plasmid backbone by gel electrophoresis, excised from the gel and purified using GenecleanTM Spin Kit. The isolated fragment was then cloned into the ⁇ co I and EcoR I sites of insert donor pE ⁇ TR 11 (Life Technologies). Briefly, pE ⁇ TR 11 was digested with ⁇ co I and EcoR I, purified by gel excision and dephosphorylated with shrimp alkaline phosphatase (Roche).
  • the lac Z fragment was then ligated to the pENTR 11 plasmid in vitro using T4 DNA ligase (Roche) and transformed into E.coli DH10B electrocompetent cells (Life Technologies). Following overnight growth on kanamycin plates, a pENTR 11 clone containing lacZ was identified by restriction digest of plasmid DNA. Thus this clone contained the lacZ coding sequence flanked by 1 bacteriophage attLl and attL2 sites.
  • vector donor comprising the following was prepared:
  • This vector donor was incubated together with insert donor (pE ⁇ TR 11 / lacZ) in a buffer containing recombination mediator proteins Lit, HF, and Xis (LR clonaseTM, Life
  • the resultant D ⁇ A was transformed into E.coli DH10B electrocompetent cells and plated out on ampicillin plates. 15 colonies were selected from the several thousand present and analysed by restriction digest of prepared plasmid D ⁇ A. Of these, 14
  • adenoviral lacZ plasmid D ⁇ A was digested with Pac I to remove the plasmid backbone and transfected into HEK 293 cells using LipofectamineTM (Life Technologies). After 12 days incubation, viral growth was apparent and virus was harvested from cells lysates.

Abstract

La présente invention concerne un système à deux constituants qui permet de cloner in vitro un polynucléotide hétérologue sous forme d'ADN adénoviral. Le premier constituant est un donneur d'insert comprenant un polynucléotide hétérologue codant un polypeptide hétérologue. Le deuxième constituant est un donneur de vecteur comprenant un génome d'adénovirus et une cassette d'expression. Le donneur d'insert et le donneur de vecteur sont adaptés à une recombinaison à spécificité de site effectuée à l'aide de sites de recombinaison provenant du phage lambda pour insérer le polynucléotide hétérologue dans la cassette d'expression capable de former un clone d'expression d'adénovirus in vitro en présence d'au moins une protéine médiateur de recombinaison appropriée. Cette invention concerne également un procédé de production d'adénovirus de recombinaison et l'utilisation des premier et deuxième constituants dans ce même procédé.
PCT/GB2001/004916 2000-11-10 2001-11-06 Vecteur WO2002038783A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2002542098A JP2004513638A (ja) 2000-11-10 2001-11-06 ベクター
EP01980719A EP1337656A1 (fr) 2000-11-10 2001-11-06 Vecteur
AU2002212507A AU2002212507A1 (en) 2000-11-10 2001-11-06 Vector
US10/416,128 US20040048381A1 (en) 2000-11-10 2001-11-06 Vector

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB0027501.6 2000-11-10
GBGB0027501.6A GB0027501D0 (en) 2000-11-10 2000-11-10 Vector
US25292700P 2000-11-27 2000-11-27
US60/252,927 2000-11-27

Publications (1)

Publication Number Publication Date
WO2002038783A1 true WO2002038783A1 (fr) 2002-05-16

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2001/004916 WO2002038783A1 (fr) 2000-11-10 2001-11-06 Vecteur

Country Status (4)

Country Link
EP (1) EP1337656A1 (fr)
JP (1) JP2004513638A (fr)
AU (1) AU2002212507A1 (fr)
WO (1) WO2002038783A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5888732A (en) * 1995-06-07 1999-03-30 Life Technologies, Inc. Recombinational cloning using engineered recombination sites
US5922576A (en) * 1998-02-27 1999-07-13 The John Hopkins University Simplified system for generating recombinant adenoviruses
US6110735A (en) * 1994-12-01 2000-08-29 Transgene, S.A. Method for the preparation of a viral vector by intermolecular homologous recombination
WO2000052187A2 (fr) * 1999-03-05 2000-09-08 Merck & Co., Inc. Systeme evolue destine a la creation de vecteurs d'adenovirus

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6110735A (en) * 1994-12-01 2000-08-29 Transgene, S.A. Method for the preparation of a viral vector by intermolecular homologous recombination
US5888732A (en) * 1995-06-07 1999-03-30 Life Technologies, Inc. Recombinational cloning using engineered recombination sites
US5922576A (en) * 1998-02-27 1999-07-13 The John Hopkins University Simplified system for generating recombinant adenoviruses
WO2000052187A2 (fr) * 1999-03-05 2000-09-08 Merck & Co., Inc. Systeme evolue destine a la creation de vecteurs d'adenovirus

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AOKI K ET AL: "EFFICIENT GENERATION OF RECOMBINANT ADENOVIRAL VECTORS BY CRE-LOX RECOMBINATION IN VITRO", MOLECULAR MEDICINE, BLACKWELL SCIENCE, CAMBRIDGE, MA, US, vol. 5, April 1999 (1999-04-01), pages 224 - 231, XP000946596, ISSN: 1076-1551 *
BERNARD P: "New ccdB positive-selection cloning vectors with kanamycin or chloramphenicol selectable markers", GENE, ELSEVIER BIOMEDICAL PRESS. AMSTERDAM, NL, vol. 162, no. 1, 30 August 1995 (1995-08-30), pages 159 - 160, XP004042069, ISSN: 0378-1119 *
CHARTIER C ET AL: "Efficient generation of recombinant adenovirus vectors by homologous recombination in E. coli", JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 70, no. 7, 1996, pages 4805 - 4810, XP002144833, ISSN: 0022-538X *
HE T-C ET AL: "A simplified system for generating recombinant adenoviruses", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 95, March 1998 (1998-03-01), pages 2509 - 2514, XP002144832, ISSN: 0027-8424 *

Also Published As

Publication number Publication date
AU2002212507A1 (en) 2002-05-21
EP1337656A1 (fr) 2003-08-27
JP2004513638A (ja) 2004-05-13

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