WO2000072887A1 - Une nouvelle lignee cellulaire d'encapsidation pour le sauvetage, la production et le titrage des vecteurs d'amplicons adenoviraux a haute efficacite - Google Patents

Une nouvelle lignee cellulaire d'encapsidation pour le sauvetage, la production et le titrage des vecteurs d'amplicons adenoviraux a haute efficacite Download PDF

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Publication number
WO2000072887A1
WO2000072887A1 PCT/US2000/014914 US0014914W WO0072887A1 WO 2000072887 A1 WO2000072887 A1 WO 2000072887A1 US 0014914 W US0014914 W US 0014914W WO 0072887 A1 WO0072887 A1 WO 0072887A1
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vector
adenovirus
helper
gene
episome
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PCT/US2000/014914
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WO2000072887A9 (fr
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Valeri A. Krougliak
Randy C. Eisensmith
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Mount Sinai School Of Medicine
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Publication of WO2000072887A9 publication Critical patent/WO2000072887A9/fr

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    • 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
    • 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
    • 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
    • C12N2710/10351—Methods of production or purification of viral material
    • C12N2710/10352—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • 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
    • C12N2800/10—Plasmid DNA
    • C12N2800/108—Plasmid DNA episomal 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
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C12N2830/003—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor tet inducible
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/38—Vector systems having a special element relevant for transcription being a stuffer
    • 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

  • This invention pertains to methods for the efficient, large-scale, helper virus-free
  • Recombinant adenovirus vectors are a highly efficient means of transferring
  • adenovirus genes into a wide variety of cell types in vivo. Although the adenovirus has a natural
  • adenoviral vectors have quite a broad host cell range.
  • this vector system has two limitations that have prevented its
  • the first is the transient expression
  • vectors is largely transient in other tissues. For example, although 100% of hepatocytes
  • adenovirus vector expressing ⁇ -galactosidase only 0.5% to 10% of hepatocytes were still
  • El -deleted adenovirus vectors is primarily a consequence of a cytotoxic T cell-mediated
  • immunosuppressive agent cyclosporme A can significantly prolong transgene expression
  • second generation adenovirus vectors are less toxic and less immunogenic
  • a second drawback of the present vector systems is that they are contaminated
  • Such responses may lead to a
  • adenovirus vector modification is the creation of a so-called
  • helper viruses or plasmid co-transfection to provide the necessary virus proteins in
  • the present invention describes a method of producing
  • helper episome comprising an adenovirus deficient genome
  • the vector packaging the vector into virions, and recovering the virions.
  • the present invention describes a system for the helper virus
  • the vector lacks the adenovirus DBP gene, and the vector comprises the adenovirus DBP and pTP genes, and where the packaged vectors are contain substantially reduced levels of helper
  • the present invention describes a method of replicating
  • helper virus comprising the steps of amplifying and packaging the vector in a cell with a
  • helper virus capable of replication and providing helper function, where the helper virus
  • the present invention describes a cell line designated
  • VK128-3 which was deposited with the NTCC Accession No. PTA-154.
  • Figure 1 is a schematic representation showing the construction of plasmid
  • pNK9-l which is the initial skeleton of the helper episome.
  • Figure 2 is a schematic representation of the construction of plasmid pNK-A2
  • Figure 3 is a schematic representation of the construction of plasmid pNK-A3
  • Figure 4 is a schematic representation of the construction of plasmid pNK-A7 in
  • Figure 5 is a schematic representation of the construction of plasmid pVNK-A22-
  • Figure 6 is a schematic representation of the construction of plasmid pAVec4-2
  • ITRs inverted terminal repeats
  • genomic DNA was also inserted into the plasmid to optimize the size of the resulting
  • adenovirus vector for packaging into mature virions.
  • Figure 7 is a schematic representation of the construction of plasmid pAVec5 in
  • promoter was inserted into pAVec4-2 to form the plasmid pAVec5.
  • Figure 8 is a schematic representation of the construction of plasmid pVK71
  • Figure 9 is a schematic representation of the construction of pAVecl2 which
  • Figure 10 is a schematic representation of the construction of the pVNK22-
  • Figure 11 is a schematic representation of the pAVecl2-l vector that is
  • Figure 12 is a schematic representation of the construction of the pAVecl ⁇ and
  • Figure 13 is a schematic representation of the construction of the pAVecl ⁇ and
  • the adenovirus vector is one of the most efficient vehicles for in vivo gene
  • helper virus in the vector preparations may elicit significant host immune responses that
  • helper virus in the system or less than about 0.1% helper virus, based on the
  • pTP is the precursor of the terminal protein.
  • IRES internal ribosomal entry sites
  • TRE tetracycline responsive promoter
  • DBP DNN binding protein
  • Cre Cre recombinase
  • EBV replicative elements refers to the Epstein-Barr virus EB ⁇ A-1
  • antibiotic resistance gene refers to DNA that confers cellular
  • antibiotics such as hygromycin or neomycin, for example.
  • binding DNA refers to DNA which does not encode for any protein or
  • stuffer DNA has regulatory function.
  • stuffer DNA is intron DNA.
  • the stuffer DNA is intron DNA.
  • helper episome refers to a circular episomal copy of a modified
  • deleted viral genes include El, E3, E4, pTP, and DBP.
  • a helper-independent vector system has been designed that allows for the
  • the system consists of two components: a packaging cell line and a complementing virus vector.
  • the cell line based on 293
  • helper episomes are designated as pVNK-N22-l, pV ⁇ K-N24-2, pV ⁇ K-
  • pVNK-22-l ⁇ DBP helper episomes can be stably transformed into 293-Tet-On cells
  • transactivator protein (Clontech).
  • episomes can be stably transformed into pTP40 cells, which are 293 cell derivatives that
  • Example 1 express both pTP and tTA. The construction of these episomes is taught in Example 1.
  • the second component of the system contains all necessary cis
  • the pAVecl ⁇ vector can be
  • the pAVecl7 vector can be used with the pTP40 cells.
  • the pNVecl ⁇ and pAVecl7 vectors can be further modified to produce vectors that
  • expression cassettes can be modified to replacing the TRE promoter element with a bi ⁇
  • helper virus- free gutless adenovirus vectors for gene therapy.
  • the helper genome is designed such that it is constantly present
  • helper genome is a cell inside the cell at low copy number ( ⁇ 20 copies per cell). Furthermore, the helper genome is a cell inside the cell at low copy number ( ⁇ 20 copies per cell). Furthermore, the helper genome is a cell inside the cell at low copy number ( ⁇ 20 copies per cell). Furthermore, the helper genome is a cell inside the cell at low copy number ( ⁇ 20 copies per cell). Furthermore, the helper genome is a cell inside the cell at low copy number ( ⁇ 20 copies per cell). Furthermore, the helper genome is a cell at low copy number ( ⁇ 20 copies per cell). Furthermore, the helper genome is a cell inside the cell at low copy number ( ⁇ 20 copies per cell). Furthermore, the helper genome is a cell inside the cell at low copy number ( ⁇ 20 copies per cell). Furthermore, the helper genome is a cell inside the cell at low copy number ( ⁇ 20 copies per cell). Furthermore, the helper genome is a cell at low copy number ( ⁇ 20 copies per cell). Furthermore, the helper genome is a cell at low copy number ( ⁇ 20 copies per cell). Furthermore,
  • the first is by deletion of its packaging sequence.
  • the second is through the incorporation of
  • helper genome derived from an otherwise infectious
  • adenovirus plasmid lacking the adenovirus pTP gene, which is absolutely essential for
  • pTP is introduced into the cell, expression of pTP from the vector will trigger replication of
  • helper genome and expression of adenoviral genes which in turn will permit replication
  • the helper genome is integrated into the genome of the
  • the integration method improves the long-term stability of the system and allows
  • helper episome would contain an bicistronic expression cassette containing
  • the adenovirus vector that is produced in this packaging cell line consists of a
  • vector in its initial embodiment is the pTP gene.
  • the first is that the vectors
  • the decreased helper virus levels are a significant benefit of the system of the present invention. Most (if not all) current production systems
  • a second advantage is that this production system can be easily expanded
  • the cell line can fully support replication of the virus vector
  • diabetes or other acquired diseases this vector system is currently without equal.
  • the system disclosed herein has numerous uses, such as to prevent heart attacks, treat inoperable
  • cancers reduce cholesterol, and reverse atherosclerosis among other possible applications.
  • nucleic acid manipulations used in practicing the present invention
  • Example 1 Construction of the helper episome.
  • Rous sarcoma virus LTR promoter was opened between the Rous sarcoma virus LTR promoter and the SV40
  • EGFP fluorescent protein
  • This plasmid (pREP-EGFP) was then linearized by Xbal digestion and a fragment
  • the packaging signal sequence obtained by Bsgl/SgrAI digestion of the plasmid pFG140
  • helper episome For the helper episome to function as intended, it must contain a significant portion
  • the complementing adenoviral amplicon vector To prevent replication of the episome as an
  • adenovirus the gene encoding the precursor of the terminal protein (pTP) was altered by the
  • Ad type 5 genome (ATCC# VR-5), containing the pTP gene, was subcloned into the
  • helper episome is shown in Firgures 3 and 4.
  • plasmid pNEB193 New England Biolabs, Inc., Beverly MA
  • PNK-A3 was further modified through the addition of a fragment of the adenoviral
  • pTP gene contained in pNK-A7.
  • the resulting plasmid was designated pNK-A8- ⁇ pTP.
  • Epstein-Barr virus e.g. EBNA-1 expression cassette and oriP
  • Ascl/Hindlll adaptor consisting of the following two oligonucleotides: 5'-
  • oligonucleotides 5'-CTAGAGGGGGGACGT-3' and 5'-CCCCCCT-3' was constructed and
  • the pE2Al plasmid was partially digested with Dr ⁇ l and completely digest with-4 ⁇ tHto remove nucleotides 22445 through 23871 in the Ad5 genome, to remove
  • the protein expressed from this gene also contains an
  • pVNK22-l ⁇ DBP and pVNK24-2 ⁇ DBP are identical except for that in the pVNK24-
  • transformants in which the episome is being maintained by replication can be positively expressed
  • transformed cells further confirms the presence of the episome.
  • Example 3 Method for the construction of an adenovirus amplicon vector that can be
  • the adenovirus cis elements necessary for virus replication and packaging are
  • Plasmid pNC-1.2 which was constructed by inserting into
  • the DNA can be and was of no specific source or
  • elongation factor l ⁇ (EFla) promoter excised from plasmid pAVec2, which was
  • adenovirus pIX gene derived in part from the commercially- available plasmid pdElsplB
  • pIX genes to be introduced into the adenoviral amplicon vector.
  • pAVec5 was digested with Nhel and an Spel fragment
  • the 1.6 kb Ad5 DBP gene was cloned by PCR, using
  • the DBP gene was then inserted into the Nhel site an pIRES (Clontech) plasmid in front of
  • pAVecl2-l was subcloned into the Xbal site downstream of the IRES sequence in
  • Bglll-Xhol adapter consisting of the following two
  • oligonucleotides 5'-TCGAGGGATCGATGGA-3' and 5'-GATCTCCATCGATCCC-3'.
  • inducible promoter the DBP gene, the IRES sequence, the pTP gene and the SV40
  • polyadenylation signal was excised from pTRE-DBP/IRES/pTP by digestion with Clal.
  • the plasmid pAVecl7 which could be rescued as an adenovirus vector if transfected
  • Example 4 DNA transfection. rescue, propagation, and titration of the gutless adenoviral
  • Mammalian VK- 128-3 cells (293 cells stably transformed with the helper episome
  • pVNK-A22-l were grown at 37 °C in 60 mm dishes to approximately 70-80% confluence.
  • the growth media (90 % MEM + 10% Fetal Bovine Serum + 1%
  • PBS phosphate-buffered saline
  • Gibco fresh serum-free MEM
  • the purified plasmid DNA was transfected into VK- 128-3 cells using a standard
  • the cells were harvested and a crude lysate of the transfected cells was prepared.
  • VK-128-3 cells grown in 60-mm dishes were infected by 0.1 ml
  • adenoviral vector produced by this method.
  • recombinant adenoviruses improves transgene persistence and decreases inflammatory
  • a new adenoviral vector Replacement of all viral coding sequences with 28 kb of DNA
  • adenovirus type 5 precursor terminal protein adenovirus type 5 precursor terminal protein. Virology 221, 172-179.
  • helper virus-dependent adenovirus vector partial purification of a helper virus- dependent adenovirus vector.
  • helper-dependent adenovirus vector system removal of helper virus by Cre-mediated
  • adenovirus vector results in improved in vivo gene expression and decreased toxicity
  • lymphocytes to viral antigens destroy hepatocytes in mice infected with El -deleted
  • fibrosis transmembrane conductance regulator is recognized by hsp70 and degraded in a pre-

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Abstract

La présente invention concerne un procédé de production de vecteur viral d'amplicons adénoviraux ne présentant plus de gènes viraux à teneur sensiblement réduite de virus auxiliaire. L'invention concerne également un système de réplication indépendante pour le virus auxiliaire et l'encapsidation des vecteurs adénoviraux ne présentant plus de gènes viraux. Enfin, l'invention concerne une lignée cellulaire pour ledit système.
PCT/US2000/014914 1999-05-28 2000-05-26 Une nouvelle lignee cellulaire d'encapsidation pour le sauvetage, la production et le titrage des vecteurs d'amplicons adenoviraux a haute efficacite WO2000072887A1 (fr)

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AU54513/00A AU5451300A (en) 1999-05-28 2000-05-26 A novel packaging cell line for the rescue, production and titration of high-capacity adenovirus amplicon vectors

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US13648199P 1999-05-28 1999-05-28
US60/136,481 1999-05-28

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001034825A1 (fr) * 1999-11-10 2001-05-17 Genzyme Corporation Lignee cellulaire de la proteine preterminale/adn polymerase comme lignee cellulaire d'enrobage pour des vecteurs adenoviraux minimaux
WO2008112540A2 (fr) * 2007-03-09 2008-09-18 Vectorlogics, Inc. Cellules pour la production de protéines et de vecteurs adénoviraux
WO2009147271A2 (fr) 2008-06-04 2009-12-10 Proyecto De Biomedicina Cima, S.L. Système d'empaquetage d'adénovirus de grande capacité

Citations (2)

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Publication number Priority date Publication date Assignee Title
US5830727A (en) * 1995-11-18 1998-11-03 Human Gene Therapy Research Institute Herpes simplex virus amplicon mini-vector gene transfer system
US5869035A (en) * 1996-11-13 1999-02-09 Human Gene Therapy Research Institute Methods and compositions for inducing complement destruction of tissue

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5830727A (en) * 1995-11-18 1998-11-03 Human Gene Therapy Research Institute Herpes simplex virus amplicon mini-vector gene transfer system
US5869035A (en) * 1996-11-13 1999-02-09 Human Gene Therapy Research Institute Methods and compositions for inducing complement destruction of tissue

Non-Patent Citations (2)

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Title
CHEN ET. AL.: "Persistence in muscle of an adenoviral vector that lacks all viral genes", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, vol. 94, March 1997 (1997-03-01), pages 1645 - 1650, XP002930032 *
MORSY ET. AL.: "Expanded-capacity adenoviral vectors-the helper-dependent vectors", MOLECULAR MEDICINE TODAY, January 1999 (1999-01-01), pages 18 - 24 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001034825A1 (fr) * 1999-11-10 2001-05-17 Genzyme Corporation Lignee cellulaire de la proteine preterminale/adn polymerase comme lignee cellulaire d'enrobage pour des vecteurs adenoviraux minimaux
WO2008112540A2 (fr) * 2007-03-09 2008-09-18 Vectorlogics, Inc. Cellules pour la production de protéines et de vecteurs adénoviraux
WO2008112540A3 (fr) * 2007-03-09 2008-12-04 Vectorlogics Inc Cellules pour la production de protéines et de vecteurs adénoviraux
WO2009147271A2 (fr) 2008-06-04 2009-12-10 Proyecto De Biomedicina Cima, S.L. Système d'empaquetage d'adénovirus de grande capacité
ES2330826A1 (es) * 2008-06-04 2009-12-15 Proyecto De Biomedicina, S.L Sistema para empaquetamiento de adenovirus de alta capacidad.

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