WO2001025462A1 - Production de virus associes a l'adenovirus (aav) recombinants mettant en oeuvre un adenovirus comprenant des genes rep/cap associes a l'adenovirus - Google Patents

Production de virus associes a l'adenovirus (aav) recombinants mettant en oeuvre un adenovirus comprenant des genes rep/cap associes a l'adenovirus Download PDF

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Publication number
WO2001025462A1
WO2001025462A1 PCT/US2000/026948 US0026948W WO0125462A1 WO 2001025462 A1 WO2001025462 A1 WO 2001025462A1 US 0026948 W US0026948 W US 0026948W WO 0125462 A1 WO0125462 A1 WO 0125462A1
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Prior art keywords
raav
promoter
aav
gene
cells
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PCT/US2000/026948
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English (en)
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WO2001025462A9 (fr
Inventor
Haifeng Chen
Gary Kurtzman
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Genovo, Incorporated
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Publication date
Application filed by Genovo, Incorporated filed Critical Genovo, Incorporated
Priority to AU78414/00A priority Critical patent/AU7841400A/en
Priority to CA002385823A priority patent/CA2385823A1/fr
Priority to JP2001528613A priority patent/JP2003511037A/ja
Priority to US10/089,394 priority patent/US7115391B1/en
Priority to EP00968512A priority patent/EP1222299A1/fr
Publication of WO2001025462A1 publication Critical patent/WO2001025462A1/fr
Publication of WO2001025462A9 publication Critical patent/WO2001025462A9/fr
Priority to US11/509,855 priority patent/US20070065412A1/en

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • 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
    • C12N2710/10344—Chimeric viral vector comprising heterologous viral elements for production of another viral vector
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C—CHEMISTRY; METALLURGY
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/75—Vector systems having a special element relevant for transcription from invertebrates
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
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    • 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 relates to novel adenoviruses useful in the production
  • rAAN recombinant adeno-associated virus
  • the adenovirus comprises
  • the invention also provides methods of producing
  • a recombinant virus carrying a foreign DNA insert may be used to generate a recombinant virus carrying a foreign DNA insert.
  • genes to cells where the gene may be expressed, if desired, to permit
  • non-human mammals or treatment or amelioration of diseases or genetic defects in
  • mammalian viral vectors such as those that are derived from retroviruses, adenoviruses, herpes viruses, vaccinia viruses, polio viruses, adeno-
  • hybrid viruses e.g., hybrid adenovirus- AAV, see U.S. Pat. No. 5,856,152
  • Other methods include direct injection of DNA, biolistics, and the like.
  • Adeno-associated virus (AAV) systems have many advantages that
  • AAV is a helper-dependent DNA
  • parvovirus which belongs to the genus Dependovirus.
  • AAV requires helper
  • Helper functions may be
  • virus either adenovirus, herpesvirus or vaccinia. In the absence of such viruses
  • AAV establishes a latent state by insertion of its genome into a host
  • AAV has a wide
  • AAV has not been
  • AAV has a genome of about 4 7 kb in length, including inverted
  • ITRs terminal repeats
  • the AAV genome encodes two genes, rep and cap, each of which expresses a
  • Rep 68 have the same amino-terminal sequence and share the same promoter, p5,
  • Rep78 contains an exon that is alternatively spliced out in re ?68
  • Rep 52 and Rep40 have the same amino-terminal sequence and share the pl9
  • virion capsid Cap gene transcription is driven by the p40 promoter See Fig
  • AAV genome are the ITRs In certain AAV genomes, the ITRs are 145 nucleotides
  • the first 125 bp of which are capable of forming Y- or T- shaped duplex
  • the ITRs represent the minimal sequence required for replication, rescue, packaging, and integration of the AAV
  • the AAV provirus functions into the cell, such as by infection with a helper virus, the AAV provirus is
  • the rescued AAV genomes are packaged into preformed protein capsids (icosahedral symmetry approximately 20 nm in diameter) and
  • a rAAV capable of delivering the transgene to target host cells.
  • the deleted rep and cap sequences are supplied to the host cells by other viruses or
  • helper functions usually are provided by helper viruses (either wildtype or crippled viruses), plasmids containing the helper virus functions or physical
  • Rep proteins a result of replacing the native p5 promoter with a strong promoter, such as the human immunodeficiency virus long terminal repeat
  • 5,837,484 states that the p5 promoter should be replaced by a strong constitutive promoter or inducible promoter, such as the metallothionein promoter, in order to
  • (rAAV) vectors comprises co-transfecting eukaryotic cells with a plasmid
  • helper virus e.g., adenovirus or
  • rcAAV comprising rep and cap flanked by ITRs, is produced when the rep and cap genes recombine with the ITRs flanking the transgene which results in deletion of
  • a second method that has been used to produce rAAV involves co-
  • the cis plasmid carries the transgene and ITRs (the cis plasmid), a second plasmid encodes the rep and cap genes (the trans plasmid), and the third plasmid encodes the helper virus
  • adenoviral genes such as Ela, Elb, E2a and E4 (the helper plasmid).
  • a third method involves the use of a packaging cell line such as one
  • the packaging cell line may be transfected with a cis
  • plasmid comprising the transgene and ITRs, and infected by wild-type adenovirus
  • a hybrid Ad/ AAV in which a hybrid Ad vector carries the cis
  • a fourth method is provided by a prophetic example in U.S. Pat. No.
  • the method involves using a recombinant adenovirus in which the rep and cap genes of AAV replace a part of the adenovirus genome not essential for
  • an AAV/EBV plasmid vector comprising an
  • rAAV genome is introduced into a cell to produce an rAAV producer cell It is
  • rep gene is driven by its native p5 promoter or by a strong inducible promoter
  • the recombinant adenovirus comprising rep and cap is then
  • the instant invention provides an alternative production method that
  • the invention provides a novel adenovirus vector comprising rep and
  • cap genes thus providing AAV rep and cap and adenovirus helper functions in one
  • the native AAV p5 promoter upstream of rep is removed and replaced with a minimal promoter or with
  • AAV ITRs may be established in the host cell by stable integration into the host
  • cell chromosome secondary infection with an adenovirus or other viral vector
  • rep operably linked to a
  • an adenovirus The adenovirus is deleted in El or E3 alone, or a combination of
  • the adenovirus vector is further deleted in E4 In
  • rep sequences may be inserted in E4, while upstream of these rep
  • cap is inserted along with the rep gene into the adenoviral vector
  • the adenoviral vector comprising the minimal
  • promoter or promoterless rep is used in a method to produce rAAV.
  • the host cell is supplied with an rAAV genome, and the adenovirus comprising the minimal promoter or
  • promoterless rep is infected into the cell.
  • host cell is either simultaneously or sequentially co-infected with two adenoviruses, wherein one adenovirus comprises cap and rep driven from a minimal promoter or
  • the other adenovirus comprises an rAAV
  • an adenovirus comprising cap and rep
  • the method is one in
  • the host cell may stably express those
  • adenoviral sequences that are deleted from the adenovirus comprising rep and cap
  • a cell line such as 293 cells, which express El, 84-31 cells, which
  • helper virus is co-infected into the host cell
  • this adenovirus could supply the deleted adenoviral
  • the recombinant virus in another embodiment of this invention, the recombinant virus
  • carrying the rep gene may be any virus in which rep interferes with its replication
  • the recombinant viral vector comprises a rep gene in which the
  • Fig. 1 Construction of recombinant shuttle plasmids
  • Fig. 2A Genome of the parental E1/E3 deleted adenovirus.
  • FIG. 2B Schematic diagrams of the recombinant adenoviruses Ad-
  • FIG. 3A Schematic diagram of the rep-cap insert in the El locus of
  • Ad-p5-RC or Ad-HSP-RC showing the location of PCR primers relative to the viral
  • Fig. 3B Ethidium bromide stained agarose gel of PCR products
  • Lanes 1, 3, 5, 7 are PCR
  • Lanes 2, 4, 6, 8 are PCR products from viral DNA of Ad-HSP-RC.
  • M 1 kb DNA ladder size marker (Gibco BRL).
  • Fig. 6. Time-course study of rAAV production after co-infection of 293 cells with Ad-HSP-RC and Ad-AAV-LacZ.
  • Fig. 7. Multiplicity of infection study of rAAV production after co-
  • Ad-AAVLacZ infection of 293 cells lane 3
  • lane 5 Ad-p5-RC and Ad-AAV ⁇
  • Fig. 9 A. Ethidium bromide stained agarose gel of Hirt DNA samples
  • FIG. 9B Southern blot analysis of the gel shown in Fig. 9 A
  • Lane 1 lacZ DNA fragment as a positive
  • FIG. 10 Western blot analysis of Rep and Cap protein expression in
  • the instant invention relates to a novel adenoviral vector and a
  • vector comprises a rep gene in which the native AAV p5 promoter upstream of the
  • Rep78 and Rep68 are produced at much
  • host cells are infected with an adenovirus vector comprising a rep gene that lacks any promoter Although the exact amounts of Rep78 and Rep68
  • Rep68 protein levels would be expressed from this recombinant adenovirus at much lower levels than Rep52 and Rep40 or at levels much lower than that expressed by wildtype AAV during infection In one embodiment of the invention, the total amount of Rep78 and
  • Rep68 protein is less than 80%, more preferably less than 50%, of the total amount
  • the total amount of Rep78 and Rep68 is less than 25% of the total
  • the total amount of Rep78 and Rep68 is less than 15%
  • Rep52 and Rep40 produced in the infected cells.
  • promoterless rep gene demonstrate that the infected host cells also produce rAAV.
  • the instant invention demonstrates that adenoviral vectors
  • adenoviral vectors comprising a rep gene with a minimal promoter or no promoter are stably propagated in host cells. See Example 4 and Figs. 3 A
  • Example 4 demonstrates that Ad-p5-RC, which is an adenovirus
  • Ad-HSP-RC which contains a
  • HSP minimal heat shock protein promoter
  • invention is one which is stable upon propagation in a defined host cell system, such
  • Ad-AAV-LacZ does not contain LacZ DNA sequences in AAV RF DNA.
  • 293 cells express Rep and Cap proteins when co-infected with Ad-HSP-
  • LacZ while replicating rAAV is observed in 293 cells co-infected with Ad-HSP-RC and Ad-AAV-LacZ See Example 8 and Figs 8, 9A and 9B Similarly, sufficient
  • adenovirus vector comprising rep sequences downstream of no promoter
  • a “recombinant adeno-associated virus (rAAV) genome” comprises
  • viral genome may be wild type or may
  • transgene may be regulated in cis or in
  • the rAAV genome comprises a transgene flanked
  • the rAAV genome of the invention may be expressed as AAV inverted terminal repeats (ITRs).
  • ITRs AAV inverted terminal repeats
  • Ad/ AAV Ad/ AAV
  • the rAAV genome may be introduced into a host cell by any route known in the art
  • the rAAV genome can be expressed transiently or stably in the host cell.
  • a “recombinant adeno-associated virus” or “rAAV” is the AAV
  • the rAAV preferably comprises
  • the rAAV comprising a transgene is capable of transducing
  • a “flanking element” or “flanking nucleic acid” is a nucleic acid
  • flanking elements of AAV are
  • inverted terminal repeats Flanking elements may be the naturally-occurring
  • a “transgene” is a nucleic acid sequence that is to be delivered or
  • a transgene may encode a protein, peptide or
  • polypeptide that is useful as a marker, reporter or therapeutic molecule.
  • transgene also may be a selection gene, such as one for antibiotic resistance.
  • transgene may also encode a protein, polypeptide or peptide that is useful for
  • transgene may not encode a protein but rather be
  • RNA molecules used as a sense or antisense molecule, ribozyme or other regulatory nucleic acid to modulate replication, transcription or translation of a nucleic acid to which it is
  • “Expression control sequences” are nucleic acid sequences that regulate the expression of a gene by being operably linked to the gene of interest.
  • Expression control sequences include appropriate transcription initiation,
  • RNA processing signals such as splicing and polyadenylation signals
  • sequences that stabilize cytoplasmic RNA processing signals such as splicing and polyadenylation signals
  • sequences that enhance protein stability are sequences that enhance protein stability; and when desired, sequences that enhance protein stability.
  • a "transgene cassette” is a nucleic acid sequence comprising a
  • transgene operably linked to expression control sequences in which the transgene
  • AAV flanking sequences flanked by AAV flanking sequences.
  • flanking sequences are AAV ITRs.
  • An "adenovirus genome” is the nucleic acid molecule backbone of an
  • the adenovirus genome may contain point mutations, deletions
  • the adenovirus genome may further comprise a
  • adenovirus is an encapsidated adenovirus genome capable of binding to a mammalian cell and delivering the adenovirus genome to the cell's
  • adenovirus encompasses both recombinant and non-recombinant cells
  • adenovirus also encompasses both wildtype and mutant adenoviruses.
  • a "recombinant adenovirus” is an adenovirus which contains one or
  • adenovirus vector is a recombinant adenovirus comprising one
  • adenovirus vector is capable of binding to a
  • the foreign gene is a mammalian cell and delivering the foreign gene to the cell's nucleus.
  • genes include, without limitation, genes such as rep and cap, rAAV genomes, such as transgenes and expression control sequences, or any foreign gene that is useful in
  • locus is a site within a virus wherein a particular gene normally
  • the "adenovirus El locus" is the site at which El resides in
  • adenovirus If a foreign gene or nucleic acid is inserted into a locus, it may either
  • AAV p5 promoter or "p5 promoter” is one that is derived
  • AAV serotypes 1 to 6 AAV Serotypes 1 to 6, as well as any AAV that infects non-human species, such as avian and bovine AAV.
  • AAV Serotypes 1 to 6 AAV Serotypes 1 to 6
  • AAV that infects non-human species such as avian and bovine AAV.
  • AAV-2 directs the expression of rep78 and rep68, and is downregulated by the Rep protein, and is upregulated by certain adenoviral proteins, including El .
  • mutant p5 promoter refers to a p5
  • the p5 promoter may be
  • promoter is effectively deleted by measuring the transcription of a gene operably linked to the mutated or partially deleted p5 promoter and comparing the gene's
  • a p5 promoter is effectively deleted
  • a p5 promoter is effectively deleted when it promotes less than 25%> of wildtype p5 promoter activity. In an even more preferred embodiment, a p5 promoter is
  • a p5 promoter is effectively deleted when the rep gene to which it is operably linked is not rearranged or deleted when an adenovirus
  • a p5 promoter comprising the effectively deleted p5 promoter and rep gene is infected into a host cell, such as 293 cells.
  • a host cell such as 293 cells.
  • a p5 promoter is
  • the deleted p5 promoter produces rAAV at a high titer.
  • the titer is at least 10 2 particles per cell; preferably at least 10 3 particles per cell;
  • TU transducing units
  • a "minimal promoter” is one that essentially comprises only a TATA
  • a promoter is a nucleotide sequence that promotes the initiation of transcription at
  • a minimal promoter promotes
  • a minimal promoter is one
  • a minimal promoter is one that promotes transcription that is less than 10% of the wildtype p5 promoter, even more preferably less than 5%o,
  • a minimal promoter also may be defined by functional measures.
  • minimal promoter is one in which the rep gene to which it is operably linked is not rearranged or deleted when an adenovirus comprising the minimal promoter and rep
  • a minimal promoter is one in
  • the titer is at least 10 2 particles per cell; preferably at least
  • artificial minimal promoter may be constructed by using a sequence or a consensus
  • the activity of the minimal promoter may be measured by measuring the transcription of the artificial minimal promoter and comparing it to an
  • Drosophila heat shock protein promoter such as the Drosophila heat shock protein promoter.
  • Rep78/68 is "promoterless” or has “no promoter” when the p5
  • promoter has been deleted or effectively deleted, as defined supra, and no promoter
  • rep78/68 is promoterless when the p5
  • promoter has been deleted and is replaced by a heterologous promoter that does not promote transcription in the host cell in which the adenovirus has been infected.
  • rep78/68 would be considered promoterless if p5 were substituted by
  • rep78/68 is
  • adenoviruses types 1-46 including human adenoviruses types 1-46, chimpanzee adenoviruses, canine
  • bovine adenoviruses all available from the American Type Culture
  • ovine adenoviruses (Both et al , WO 97/06826 Al). Any of these adenoviruses may
  • adenovirus is able to infect the target host cell
  • a human adenovirus would generally be used to infect a
  • bovine adenovirus would be used to infect a bovine cell.
  • the adenoviral vector comprises the AAV rep
  • minimal promoter or promoterless AAV rep gene referred to as a minimal promoter or promoterless AAV rep gene
  • helper virus functions for rAAV production in a host cell In a preferred embodiment
  • the adenoviral vector further comprises the AAV cap gene The type
  • helper functions For instance, if the adenovirus is to be used to infect a cell line
  • the adenoviral vector could comprise rep and cap, and could also comprise those helper virus functions required in
  • adenovirus vector could express rep, cap, E2a and VAI RNA If the adenovirus is used to infect a cell line that does not express any helper functions, then the adenovirus
  • vector could comprise, at least, El (both El a and Elb) and E2a, and, optionally,
  • helper may comprise E4ORF6 and VAI RNA.
  • helper may comprise E4ORF6 and VAI RNA.
  • the recombinant adenovirus comprising the rep gene downstream of a minimal promoter or no promoter may be produced by any method known in the
  • the recombinant adenovirus of the instant invention is
  • recombinant adenovirus is produced using Cre-lox recombination (12).
  • the host cell may be co-infected with a second virus, such as
  • an adenovirus that expresses some or all of the required helper functions.
  • a second adenoviral vector comprises a transgene cassette
  • adenovirus comprising the rep gene. See, e.g., Examples 6-8.
  • Examples 6-8 See, e.g., Examples 6-8.
  • helper virus functions may be provided by any method known in the art, such as by transfection or direct injection, as discussed above.
  • Rep and Cap proteins may have a naturally
  • the rep and cap genes are derived from the same
  • rAAV Pseudotyped rAAV is desirable in cases in which the rAAV is to be administered to a patient as a gene therapy vector and there are existing neutralizing
  • antibody response may be exchanged by the cap gene from a different serotype of
  • AAV to which there is no antibody response For example, the rep gene from
  • AAV-2 may be used with the cap gene from AAV-1 to produce a pseudotyped rAAV-2, or vice-versa
  • the Rep and/or Cap proteins may have
  • mutated sequence including insertions, deletions, fragments or point mutations of particular amino acid residues, so long as the mutated Rep and/or Cap proteins retain their respective excision, replication and encapsidation functions
  • mutated Rep and/or Cap proteins retain their respective excision, replication and encapsidation functions
  • a single adenovirus comprises the
  • nucleic acid sequences encoding the Rep and Cap proteins are inserted at a
  • both rep and cap are inserted at El, E3 or
  • nucleic acid sequence encoding Rep may be in El, and the nucleic acid sequence encoding Cap may be in E4, and other combinations thereof Alternatively, a cell
  • the promoter may be any promoter that promotes only basal expression of the rep gene in a host
  • the promoter is one that essentially contains a TATA box as its only regulatory element
  • the minimal promoter is the
  • HSP Drosophila heat shock promoter
  • minimal promoter is the minimal promoter derived from the adenovirus Elb gene
  • minimal promoter is a 70 nucleotide DNA element derived from the promoter
  • the minimal pIX promoter comprises
  • the p5 promoter is deleted altogether and replaced by no promoter at all
  • adenovirus comprising a rep gene whose expression is regulated by a minimal
  • the host cell may comprise the rAAV genome stably or transiently.
  • Rep78 and Rep68 may be measured in the host cell after infection to determine if sufficiently low levels of Rep78 and Rep68 are
  • AAV requires helper functions for excision, replication and encapsidation of AAV.
  • AAV helper functions can be provided by
  • herpesvirus including herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2), cytomegalovirus (CMV) and pseudorabies virus (PRV)] or by exposure
  • HSV-1 herpes simplex virus type 1
  • HSV-2 herpes simplex virus type 2
  • CMV cytomegalovirus
  • PRV pseudorabies virus
  • rAAV production one may transfect a host cell containing an rAAV genome with a plasmid comprising rep and cap and then transfect with one or nucleic acids
  • the rAAV genome may be stably
  • the host cell may be transfected or infected into the host cell by methods known in the art. After transfecting the host cell with the nucleic acid
  • nucleic acid encodes a helper function
  • helper functions are nucleic acids
  • helper functions are
  • HSV-1 derived from adenovirus types 2 or 5, HSV-1, HSV-2, CMV or PRV.
  • helper functions are El a, Elb, E2a, E4ORF6
  • nucleic acid encodes the helper functions from the helicase-primase complex of
  • helper functions for recombinant AAV may be
  • chemical or physical agents including ultraviolet light, cycloheximide,
  • the required helper functions for production of a rAAV may be
  • helper functions may be any method known in art.
  • the helper functions may be any method known in art.
  • a vector such as a plasmid
  • the transfection or infection may be stable or transient
  • the cell line may stably express (either on an extrachromosomal episome or through integration in the cell's genome) the helper functions
  • helper functions may be expressed by the mammalian cell line
  • helper functions are transduced into
  • helper functions are transduced into the host cell by the adenovirus that comprises
  • the native helper function is the native helper function
  • helper function nucleic acids are used However, mutated helper function sequences may be used so long as they retain their helper function activity
  • CMV immediate-early may be supplied with its native promoter or may be under the regulatory control of a variety of promoters, constitutive or inducible, such as the CMV immediate-early
  • ERS expression regulatory sequences
  • AAV flanking sequences e.g., the ITRs, in place of rep and cap genes normally
  • an optional spacer or "sniffer" sequence may be inserted in order
  • the transgene cassette comprised of
  • the ERS-transgene bordered by the AAV flanking sequences may then be embedded
  • the transgene cassette may be inserted into a plasmid vector and
  • transfected into a host cell The transgene cassette may be maintained in the host
  • cell stably, either by integration into the host cell genome or as an episome, or may
  • transgene cassette e.g., Examples 3 and 6-8.
  • Each element of the transgene cassette is further
  • a transgene is a nucleic acid encoding a protein of interest; it may be
  • a gene to allow for genetic or drug selection e.g., a gene conferring resistance to
  • the transgene may be one that is
  • a transgene may be a normal gene
  • the transgene may be one that counteracts the effects of a disease, such as introduction and
  • transgene may be a gene which blocks or represses the expression of a malfunctioning, mutated, or viral gene in the patient, thereby giving rise to a
  • a transgene may also be a protective gene, such as one that
  • a transgene may also be used
  • transgene also has the following properties: correction of a defect or is beneficial for prevention of disease.
  • the transgene also has the following properties: correction of a defect or is beneficial for prevention of disease.
  • the transgene also may be one which is useful for production of
  • cystic fibrosis there are one or more mutations
  • CFTR CFTR which prevents the CFTR protein from functioning properly.
  • CFTR CFTR
  • islet cells which produce insulin, are destroyed, such that patients with this disease
  • the endogenous gene may be any other gene that can no longer synthesize insulin.
  • the endogenous gene may be any other gene that can no longer synthesize insulin.
  • the endogenous gene may be any other gene that can no longer synthesize insulin.
  • the endogenous gene may be any other gene that can no longer synthesize insulin.
  • the endogenous gene may be any other gene that can no longer synthesize insulin.
  • the endogenous gene may be any other genes
  • EPO erythropoietin
  • transgene encoding EPO
  • overexpression of a particular gene results in a disease state. For instance, overexpression of z-myc by the immunoglobulin heavy chain promoter results in
  • leukemia Transgenes may also be used for genetic immunization, i e , to elicit an
  • transgene may include a sequence from a viral, bacterial or fungal pathogen, such as
  • influenza virus human immunodeficiency virus (HIV), or mycobacterium
  • the appropriate gene for expression is one which expresses a normal gene product
  • proteins such as secreted factors, including hormones, growth factors
  • composition of the transgene sequence depends upon the
  • transgene sequence For example, one type of transgene sequence
  • reporter or marker sequence comprises a reporter or marker sequence, which upon expression produces a detectable signal.
  • reporter or marker sequences include, without limitation,
  • GFP green fluorescent protein
  • chloramphenicol acetyltransferase CAT
  • firefly luciferase eukaryotic membrane
  • proteins including, for example, CD2, CD4, CD8, the influenza
  • hemagglutinin protein and others well known in the art, to which high affinity
  • fluorescent activated cell sorting assay and immunological assays, including ELISA,
  • the transgene is luciferase
  • the rAAV gene expression may be measured by light production in a luminometer.
  • the transgene is a non-marker gene which can
  • transgene may be selected from a wide variety of gene products useful in biology
  • RNAs sense or antisense nucleic acids
  • catalytic RNAs catalytic RNAs
  • sequence is a therapeutic gene which expresses a desired corrective gene product in
  • a host cell at a level sufficient to ameliorate the disease including partial
  • the selected transgene may encode any product desirable for study.
  • transgene sequence is within the skill of the artisan in accordance with the teachings
  • the invention also includes methods of producing rAAV and
  • compositions thereof which can be used to correct or ameliorate a gene defect
  • transgene may be used to encode each subunit of the protein. This may be desirable when the size
  • DNA encoding the protein subunit is large, e.g., for an immunoglobulin or
  • the platelet-derived growth factor receptor In order for the cell to produce the multi-subunit protein, a cell would be infected with rAAV expressing each of the
  • transgene may be encoded by the same transgene. In this case, a single transgene would be encoded by the same transgene. In this case, a single transgene would be encoded by the same transgene. In this case, a single transgene would be encoded by the same transgene. In this case, a single transgene would be encoded by the same transgene. In this case, a single transgene would be encoded by the same transgene. In this case, a single transgene would be encoded by the same transgene. In this case, a single transgene would be encoded by the same transgene. In this case, a single transgene would be encoded by the same transgene. In this case, a single transgene would be encoded by the same transgene. In this case, a single transgene would be encoded by the same transgene. In this case, a single transgene would be encoded by the same transgene. In this case, a single transgen
  • IRES internal ribosome entry site
  • IRES elements can be linked to heterologous molecules
  • each open reading frame is
  • the insert size can be no greater than approximately 4 8
  • the insert size is approximately 28 kilobases
  • Useful gene products include hormones and growth and
  • differentiation factors including, without limitation, insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), calcitonin, growth hormone releasing
  • GRF thyroid stimulating hormone
  • TSH thyroid stimulating hormone
  • TRH thyrotropin-releasing hormone
  • FSH luteinizing hormone
  • CG chorionic gonadotropin
  • VEGF endothelial growth factor
  • angiopoietins angiostatin
  • endostatin endothelial growth factor
  • GCSF granulocyte colony stimulating factor
  • EPO erythropoietin
  • CTGF connective tissue growth factor
  • bFGF basic fibroblast growth factor
  • bFGF2 acidic
  • fibroblast growth factor aFGF
  • epidermal growth factor EGF
  • TGF ⁇ growth factor ⁇
  • PDGF platelet-derived growth factor
  • IGF-I and IGF-II growth factors I and II (IGF-I and IGF-II), any one of the transforming growth
  • TGF ⁇ factor ⁇
  • BMP morphogenic proteins
  • nerve growth factor nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • neurotrophins NT-3, NT-4/5 and NT-6 neurotrophins NT-3, NT-4/5 and NT-6, ciliary neurotrophic factor (CNTF), glial
  • GDNF derived neurotrophic factor
  • neurtuin neurtuin
  • persephin agrin
  • HGF hepatocyte growth factor
  • ephrins noggin
  • sonic hedgehog tyrosine hydroxylase
  • cytokines and lymphokines including, without limitation, cytokines and lymphokines such as
  • MCP-1 monocyte chemoattractant protein
  • LIF leukemia inhibitory factor
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • G-CSF G-CSF
  • M-CSF monocyte colony stimulating factor
  • Fas Fas
  • IFN interferons
  • IFN- ⁇ and IFN- ⁇ stem cell factor, flk-2/flt3 ligand.
  • immune system are also encompassed by this invention. These include, without limitations, immunglobulins IgG, IgM, IgA, IgD and IgE, chimeric
  • immunoglobulins humanized antibodies, single chain antibodies, T cell receptors, and fragments thereof
  • chimeric T cell receptors single chain T cell receptors, class I and class II MHC
  • Useful gene products also include complement regulatory proteins such as membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, CR2
  • MCP membrane cofactor protein
  • DAF decay accelerating factor
  • CR1 CR2
  • Still other useful gene products include any one of the receptors for
  • hormones the hormones, growth factors, cytokines, lymphokines, regulatory proteins and
  • receptors include flt-1, flk-1, TIE-2; the trk family of receptors such as TrkA, MuSK, Eph, PDGF receptor, EGF
  • TGF ⁇ receptors TGF ⁇ receptors, the interleukin receptors, the interferon receptors, serotonin
  • the invention encompasses receptors for
  • extracellular matrix proteins such as integrins, counter-receptors for transmembrane-bound proteins, such as intercellular adhesion molecules (ICAM-1, ICAM-2, ICAM-3 and ICAM-4), vascular cell adhesion molecules (VCAM), and
  • LDL receptor for cholesterol regulation, including the LDL receptor, HDL receptor, VLDL
  • the inventions encompasses the
  • apolipoprotein ligands for these receptors including ApoAI, ApoAIV and ApoE.
  • the invention also encompasses gene products such as steroid hormone receptor
  • useful gene products include
  • antimicrobial peptides such as defensins and maginins
  • transcription factors such as
  • IRF-1 interferon regulation factor 1
  • ETS-binding protein STAT
  • GATA-box binding proteins e.g., GATA-3
  • forkhead family of winged helix proteins e.g., GATA-3
  • transcarbamylase arginosuccinate synthetase, arginosuccinate lyase, arginase,
  • fumarylacetoacetate hydrolase phenylalanine hydroxylase, alpha- 1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor VII, factor VIII, factor
  • angiotensin converting enzyme endothelin-1, atrial natriuetic peptide, pro-
  • urokinase urokinase
  • urokinase urokinase
  • plasminogen activator plasminogen activator
  • heparin cofactor II activated protein
  • CoA carboxylase methyl malonyl CoA mutase, glutaryl CoA dehydrogenase,
  • insulin beta-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase (also referred to as P-protein), H-
  • T-protein T-protein
  • Menkes disease protein T-protein
  • tumor suppressors e.g., p53
  • CFTR fibrosis transmembrane regulator
  • lactase lactase, lipase, trypsin, gastrointestinal enzymes including chyromotrypsin, and
  • pepsin adenosine deaminase
  • ⁇ l anti-trypsin tissue inhibitor of metalloproteinases
  • TRIP hexokinases
  • glucokinase any one or more of the individual chains or types of collagen
  • elastin fibronectin
  • thrombospondin vitronectin and tenascin
  • suicide genes such as thymidine kinase and cytosine deaminase.
  • Other useful proteins include
  • galactosidase a ⁇ -1-iduronidase, iduroate sulfatase, lysosomal acid ⁇ -glucosidase,
  • sphingomyelinase hexosamina ⁇ idase A, hexominidases A and B, arylsulfatase A, acid lipase, acid ceramidase, galactosylceramidase, ⁇ -fucosidase, ⁇ -, ⁇ -
  • mannosidosis aspartylglucosaminidase, neuramidase, galactosylceramidase,
  • 6-sulfate sulfatase 6-sulfate sulfatase, arylsulfatase B, ⁇ -glucuoronidase and hexosaminidases A and B.
  • polypeptides such as chimeric or hybrid polypeptides or polypeptides having a non-
  • single-chain engineered immunoglobulins could be useful in certain immunocompromised patients.
  • Other useful proteins include
  • truncated receptors which lack their transmembrane and cytoplasmic domain.
  • truncated receptors can be used to antagonize the function of their respective
  • non-naturally occurring gene sequences include sense and antisense
  • transgenes include those that encode antigenic peptides
  • transgenes can be used for genetic immunization.
  • Useful transgenes include those that encode peptides specific for Epstein Barr virus; HIV; simian immunodeficiency
  • SIV human T-cell leukemia viruses I and II (HTLV-I and HTLV-II);
  • hepatitis A, B, C, D, E and SEN pseudorabies virus
  • rabies virus cytomegalovirus
  • respiratory syncytial virus respiratory syncytial virus
  • parainfluenza virus types 1-4 mumps virus; rubella virus; polio virus; measles virus; influenza virus types A, B and C; rotavirus; he ⁇ es
  • hantavirus denguevirus, Sindbisvirus, adenoviruses; chlamydia pneumoniae;
  • chlamydia trachomatis mycoplasma pneumoniae; mycobacterium tuberculosis;
  • the transgenes may also be directed against peptides from tumor antigens to provide immunization for tumors and cancers.
  • constitutive, inducible and/or tissue-specific ⁇ are known in the art and may be
  • expression control sequences typically include a promoter, an enhancer, such as one derived from an
  • the polyadenylation sequence generally is inserted following the
  • transgene-carrying molecule useful in the present invention may also contain an
  • intron desirably located between the promoter/enhancer sequence and the transgene
  • intron sequence is also derived from SV40 and is referred
  • IRES internal ribosome entry site
  • high-level constitutive expression will be
  • promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter/enhancer, the cytomegalovirus (CMV)
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • inducible promoters may be desired
  • Inducible promoters are those which are regulated by exogenously supplied
  • MT zinc-inducible metallothionine
  • Dex dexamethasone
  • MMTV mammary tumor virus
  • T7 polymerase promoter the T7 polymerase promoter system
  • promoters which may be useful in this context are those which are regulated by a
  • the native promoter for the transgene or
  • nucleic acid sequence of interest will be used.
  • the native promoter may be any promoter
  • control elements such as enhancer elements, polyadenylation sites or Kozak
  • consensus sequences may also be used to mimic the native expression
  • the recombinant viral genome comprises a
  • transgene operably linked to a tissue-specific promoter For instance, if expression in skeletal muscle is desired, a promoter active in muscle may be used These include the promoters from genes encoding skeletal ⁇ -actin, myosin light chain 2A,
  • promoters that are tissue-specific are known for liver [albumin, Miyatake et al J Virol , 71 5124-32 (1997), hepatitis B virus core
  • lymphocytes [CD2, Hansal et al , J Immunol . 161 1063-8 (1998), lmmunoglobulin heavy chain, T cell receptor ⁇ chain], neuronal
  • NSE neuroon-specific enolase
  • host cell of choice may be selected by one of skill in the art using the guidance
  • adenovirus vector In one embodiment, one may insert a recombinant viral genome
  • the number of copies of the transgene in the cell may be monitored by
  • expression may be monitored by Western blotting, immunohistochemistry, ELISA,
  • RIA tests of the transgene' s gene product's biological activity, either in vitro or in vivo, or tests for correction or amelioration of a genetic defect.
  • AAV ITRs consist of repeated sequences
  • the AAV ITRs are required for replication, excision and encapsidation of both wild type and recombinant AAV virions.
  • the ITRs flank the
  • rAAV is rescued from the host chromosome, the ITRs excise along with the transgene and remain in flanking positions surrounding the rescued DNA, in a form
  • the ITRs may be derived from any one of the
  • the rAAV comprises a selected transgene operably linked to expression regulatory sequences and AAV flanking elements
  • Any type of mammalian cell that can be adapted to cell culture may be any type of mammalian cell that can be adapted to cell culture.
  • a host cell to produce the recombinant viral genome
  • a host cell to produce the recombinant viral genome
  • a host cell to produce the recombinant viral genome
  • cell used in this invention is one that may be infected by the adenovirus vector of
  • Appropoate host cells include, without limitation, CHO, BHK,
  • MDCK and vaoous muone cells e g , 10T1/2 and WEHI cells, African green
  • monkey cells such as VERO, COS 1, COS 7, BSC 1, BSC 40, and BMT 10, and
  • human cells such as WI38, MRC5, A549, human embryonic retinoblast (HER),
  • HTK human embryonic kidney
  • HEL human embryonic lung
  • appropoate cells include 293 cells (human embryonic
  • kidney cells that express adenoviral Ela and Elb proteins), 911 or PER C6 cells kidney cells that express adenoviral Ela and Elb proteins), 911 or PER C6 cells
  • B50 cells HeLa cells that express AAV rep and cap, see PCT US98/19463
  • 3T3 cells mouse embryonic fibroblast cell line
  • NIH3T3 cells subline of 3T3 cells
  • HepG2 HepG2
  • infectious rAAV One may then compare the titer of infectious rAAV produced in
  • Another aspect of the instant invention is a method of producing
  • the method comprises the
  • adenovirus comprising a rep gene under the regulatory control of a minimal promoter or no promoter
  • rAAV genome is excised, replicated and encapsidated
  • the host cells may be any mammalian cell known in the art or as
  • the host cell prior to infection by the adenovirus comprising the
  • rep gene may be one that expresses one or more of the following genetic elements:
  • the host cell may comprise none of these genetic elements prior to infection by the adenovirus.
  • the genetic elements are
  • the adenovirus comprising the rep gene, other viral vectors, and/or
  • the host cells may be infected by the adenovirus by any method
  • Rep52 and Rep40 both expressed from the adenoviral pl9 promoter, and the capsid proteins, expressed from the AAV p40 promoter, are sufficient to
  • the rAAV may be purified from the supernatant produced by the host cells or from cell lysates by any method known in the art or as described
  • the method is easily scaled to industrial production because it does
  • host cells are co-infected with two different adenoviruses, one comprising rep downstream of a minimal promoter or no
  • Infection of host cells by adenovirus is highly efficient and may be easily scaled to a
  • the instantly described method produces rAAV at a high titer In a
  • the titer is at least 10 2 particles per cell, preferably at least
  • the instant invention also encompasses lysates and supernatants of host cells comprising rAAV These lysates
  • the rAAV produced by the method of this invention may be
  • the rAAV comprises at least the rAAV and a pharmaceutically acceptable carrier
  • the rAAV comprises at least the rAAV and a pharmaceutically acceptable carrier
  • the rAAV may be introduced into cells ex vivo or
  • the rAAV may be used to infect a cell in vitro, and then the cell may subsequently be introduced into a
  • the mammal e.g., into the portal vein or into the spleen, if desired.
  • the mammal e.g., into the portal vein or into the spleen
  • the spleen e.g., into the portal vein or into the spleen
  • rAAV may be administered to a mammal directly, e.g., intravenously or
  • a slow-release device such as an implantable pump, may be used
  • virus is administered to a cell.
  • the specific cells to be infected may be targeted by controlling the method
  • rAAV intravascular administration of rAAV to the portal vein or
  • rAAV to a liver cell may be used to facilitate targeting rAAV to a liver cell.
  • the rAAV produced by the above-described method may be
  • a suitable vehicle includes sterile saline.
  • the rAAV is administered in sufficient amounts to infect the desired
  • transgene or viral gene products in the case of a vaccine
  • the dosage may also vary depending on the condition being treated and the selected gene.
  • the dosage may also vary depending on the condition being treated and the selected gene.
  • an effective human upon the age, weight and health of the patient.
  • an effective human for example, an effective human
  • dosage of rAAV is generally in the range of from about 0.5 ml to 50 ml of saline
  • the levels of expression of the selected gene may be monitored to determine the type and frequency of dosage
  • Penicillin 50 ⁇ g/ml of streptomycin, and 10 ⁇ g/ml of neomycin (Gibco BRL).
  • Human embryonic kidney cell line 293 is obtained from ATCC (CRL 1573). 293-
  • 293-CG3 is a 293 -derived cell line carrying stably integrated copies of AAV ITRs flanking GFP as marker gene (Chen et al., unpublished data).
  • recombinant adenoviruses are propagated on 293 cells and purified through CsCl
  • Dralll site nucleotide 241, upstream of the AAV-2 p5
  • Ncol site nucleotide 4489, downstream of the polyA signal
  • nucleotide 4464 downstream of
  • HSP Drosophila melanogaster minimal heat shock protein
  • adenoviruses Ad-p5-RC and Ad-HSP-RC (Fig. 2) are generated according to standard protocols known in the art (see, e.g., Refs. 4 and 12). The recombinant adenoviruses are passaged five to six times on appropriate
  • mammalian cells to generate a stock of recombinant adenovirus that is used for
  • the monolayer is
  • PCR polymerase chain reaction
  • HC#30 (5'-CGTAACCGAGTAAGATTTGG-3', SEQ ID NO 1)
  • HC#31 (5'-ATGTTGGTGTTGGAGGTGAC-3', SEQ ID NO 2)
  • HC#32 (5 '-TGGACCAGAAATGCAAGTCC-3', SEQ ID NO 3),
  • HC#33 (5 '-AGCCTTGACTGCGTGGTGGT-3', SEQ ID NO 4),
  • HC#34 (5 '-GTACCTGT ATT ACTTGAGCA-3', SEQ ID NO 5)
  • HC#35 (5 -ACGAGTCAG GTATCTGGTGC-3', SEQ ID NO 6)
  • HC#36 (5 '-GGACTTTACTGTGGACACTA-3', SEQ ID NO 7), and HC#37 (5' -GACCC AGACT ACGCTGACGA-3 ' , SEQ ID NO 8)
  • RNase A and pronase followed by extraction with phenol-chloroform Adenoviral DNA in the supernatant is precipitated with isopropanol and dissolved in TE/RNase buffer (10 mM Tris-HCl, pH8.0, 1 mM EDTA, 20 ⁇ g/ml RNase).
  • the PCR assay is performed using the Robocycler Gradient 96
  • Ad-HSP-RC is shown to contain full length AAV-2 rep-cap
  • virus in 0.5 ml of serum-free DMEM are added to the cells. Following a 30 min.
  • Infected cells are harvested and pelleted. Cell pellets are lysed in 1 ml of lysis buffer (50 mM Tris-HCl, pH7.4,
  • lysate supernatant is collected for titration of rAAV.
  • lysate is diluted in DMEM containing 10%> FBS and heated at 56°C for 60 min to
  • TU transducing units
  • Ad-HSP-RC produced through infection of 293-CG3 cells with the recombinant Ad-HSP-RC.
  • the rAAV titer increases by increasing the multiplicity of infection (MOI) of Ad-
  • Ad-HSP-RC Ad-HSP-RC rescue and packaging into rAAV.
  • AAV vector sequences are delivered exogenously into 293 cells to determine whether the functionality of the Ad-HSP-RC recombinant.
  • Ad-HSP-RC To perform this experiment, 293 cells are seeded on 6-well plates at a density of lxlO 6 cells/well 12-15 hours later, the cells are co-infected with Ad-
  • HSP-RC and Ad-AAV-LacZ an El -deleted adenovirus containing the E. coli lacZ
  • viruses 50 to 450 particles/cell are mixed together, keeping the total inoculum at a
  • the virus mixture is diluted in 0 5 ml of serum-
  • Infected cells are harvested, lysed, and rAAV titrated as described in
  • Example 5 except that rAAV transduction is scored cells by X-gal staining
  • the rAAV transduced cells are first fixed with 0 5 ml of 0 05% glutaraldehyde for 10 min and then rinsed with 3x0 5 ml

Abstract

La présente invention concerne de nouveaux adénovirus utiles dans la production de titres élevés de virus associés à l'adénovirus recombinants (AAVr) comprenant un insert d'ADN étranger et des procédés de fabrication desdits adénovirus. Les adénovirus comportent le gène AAV rep dans lequel le promoteur p5 de rep est substitué par un promoteur minimal ou par aucun promoteur. L'invention concerne également des procédés de production de AAVr de haut niveau sous forme d'une préparation sensiblement homogène et des compositions de AAVr.
PCT/US2000/026948 1999-10-01 2000-09-29 Production de virus associes a l'adenovirus (aav) recombinants mettant en oeuvre un adenovirus comprenant des genes rep/cap associes a l'adenovirus WO2001025462A1 (fr)

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AU78414/00A AU7841400A (en) 1999-10-01 2000-09-29 Production of recombinant aav using adenovirus comprising aav rep/cap genes
CA002385823A CA2385823A1 (fr) 1999-10-01 2000-09-29 Production de virus associes a l'adenovirus (aav) recombinants mettant en oeuvre un adenovirus comprenant des genes rep/cap associes a l'adenovirus
JP2001528613A JP2003511037A (ja) 1999-10-01 2000-09-29 ＡＡＶｒｅｐ／ｃａｐ遺伝子を含むアデノウイルスを使用する組換えＡＡＶの産生
US10/089,394 US7115391B1 (en) 1999-10-01 2000-09-29 Production of recombinant AAV using adenovirus comprising AAV rep/cap genes
EP00968512A EP1222299A1 (fr) 1999-10-01 2000-09-29 Production de virus associes a l'adenovirus (aav) recombinants mettant en oeuvre un adenovirus comprenant des genes rep/cap associes a l'adenovirus
US11/509,855 US20070065412A1 (en) 1999-10-01 2006-08-25 Production of recombinant AAV using adenovirus comprising AAV rep/cap genes

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US60/157,466 1999-10-01

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002022800A2 (fr) * 2000-09-15 2002-03-21 Micromun Privates Institut Für Mikrobiologische Forschung Gmbh Cassettes d'expression et vecteurs d'adenovirus
WO2008156681A2 (fr) * 2007-06-14 2008-12-24 Yoshihiro Kawaoka Vecteurs d'adénovirus pour la production de virus de la grippe
EP2368903A1 (fr) * 2010-03-26 2011-09-28 Deutsches Krebsforschungszentrum Virus transporteur dérivé d'adénovirus pour améliorer la production de parvovirus recombinant
JP2011254821A (ja) * 2001-12-17 2011-12-22 Trustees Of The Univ Of Pennsylvania アデノ随伴ウイルス（ａａｖ）血清型８の配列
US9890365B2 (en) 2014-03-09 2018-02-13 The Trustees Of The University Of Pennsylvania Compositions useful in treatment of ornithine transcarbamylase (OTC) deficiency
TWI745285B (zh) 2015-01-20 2021-11-11 美商健臻公司 用於重組病毒粒子特徵化之分析性超速離心法
US11802273B2 (en) 2014-06-20 2023-10-31 Wisconsin Alumni Research Foundation (Warf) Mutations that confer genetic stability to additional genes in influenza viruses
US11807872B2 (en) 2019-08-27 2023-11-07 Wisconsin Alumni Research Foundation (Warf) Recombinant influenza viruses with stabilized HA for replication in eggs
US11851648B2 (en) 2019-02-08 2023-12-26 Wisconsin Alumni Research Foundation (Warf) Humanized cell line

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2689588C (fr) * 2007-06-21 2016-08-23 Alphavax, Inc. Cassettes sans promoteur pour l'expression de proteines structurales d'alphavirus
RU2705249C2 (ru) * 2013-07-12 2019-11-06 Дзе Чилдрен'З Хоспитал Оф Филадельфия Вектор aav и анализ на нейтрализующие антитела против aav (аденоассоциированного вируса)
HUE046643T2 (hu) * 2014-11-28 2021-11-29 Uniqure Ip Bv DNS szennyezõdések parvovirális virionokat tartalmazó készítményben

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996013598A2 (fr) * 1994-10-28 1996-05-09 The Trustees Of The University Of Pennsylvania Virus hybride adenovirus-aav et ses procedes d'utilisation
WO1998006864A2 (fr) * 1996-08-15 1998-02-19 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Regulation spatiale et temporelle de l'expression genique au moyen d'un promoteur proteique du stress associe a une source de chaleur locale
US5780280A (en) * 1990-10-30 1998-07-14 Rhone-Poulenc Rorer Pharmaceuticals, Inc. Recombinant adeno-associated virus vectors
WO1998046728A1 (fr) * 1997-04-14 1998-10-22 Cell Genesys, Inc. Procede d'amelioration de l'efficacite d'un produit d'aav recombine

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5780280A (en) * 1990-10-30 1998-07-14 Rhone-Poulenc Rorer Pharmaceuticals, Inc. Recombinant adeno-associated virus vectors
WO1996013598A2 (fr) * 1994-10-28 1996-05-09 The Trustees Of The University Of Pennsylvania Virus hybride adenovirus-aav et ses procedes d'utilisation
WO1998006864A2 (fr) * 1996-08-15 1998-02-19 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Regulation spatiale et temporelle de l'expression genique au moyen d'un promoteur proteique du stress associe a une source de chaleur locale
WO1998046728A1 (fr) * 1997-04-14 1998-10-22 Cell Genesys, Inc. Procede d'amelioration de l'efficacite d'un produit d'aav recombine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KYÖSTIÖ S.R.M. ET AL.: "Negative regulation of the Adeno-Associated Virus (AAV) P5 promoter involves both the P5 Rep binding site and the consensus ATP-binding motif of the AAV Rep68 protein.", J. VIROL., vol. 69, no. 11, November 1995 (1995-11-01), pages 6787 - 6796, XP002158144 *
WANG X.S. & SRIVASTAVA A.: "Rescue and autonomous replication of Adeno-Associated Virus Type 2 genomes containing Rep-binding site mutations in the viral P5 promoter.", J. VIROL., vol. 72, no. 6, June 1998 (1998-06-01), pages 4811 - 4818, XP002158145 *

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