WO2001025462A9 - 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

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Publication number
WO2001025462A9
WO2001025462A9 PCT/US2000/026948 US0026948W WO0125462A9 WO 2001025462 A9 WO2001025462 A9 WO 2001025462A9 US 0026948 W US0026948 W US 0026948W WO 0125462 A9 WO0125462 A9 WO 0125462A9
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WO
WIPO (PCT)
Prior art keywords
raav
promoter
aav
gene
cells
Prior art date
Application number
PCT/US2000/026948
Other languages
English (en)
Other versions
WO2001025462A1 (fr
Inventor
Haifeng Chen
Gary Kurtzman
Original Assignee
Genovo Inc
Haifeng Chen
Gary Kurtzman
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genovo Inc, Haifeng Chen, Gary Kurtzman filed Critical Genovo Inc
Priority to AU78414/00A priority Critical patent/AU7841400A/en
Priority to CA002385823A priority patent/CA2385823A1/fr
Priority to EP00968512A priority patent/EP1222299A1/fr
Priority to US10/089,394 priority patent/US7115391B1/en
Priority to JP2001528613A priority patent/JP2003511037A/ja
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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    • C12N2710/00—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
    • C12N2710/10344—Chimeric viral vector comprising heterologous viral elements for production of another viral vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • 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
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • 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
    • C12N2830/75—Vector systems having a special element relevant for transcription from invertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
  • rAAV 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-viral viruses
  • hybrid viruses e.g., hybrid adenovirus-AAV, see U.S. Pat. No.
  • 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 function in order for a productive infection to occur. 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 polypeptides (Rep78, Rep68, Rep52, and Rep40) are
  • Rep68 have the same amino-terminal sequence and share the same promoter, p5,
  • Rep78 contains an exon that is alternatively spliced out in rep ⁇ S.
  • Rep 52 and Rep40 have the same amino-terminal sequence and share the pl9
  • Cap proteins (VPl, VP2, and VP3) form
  • Cap gene transcription is driven by the p40 promoter. See Fig.
  • AAV genome are the ITRs.
  • 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
  • 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
  • rAAV vectors comprises co-transfecting eukaryotic cells with a plasmid containing rAAV sequences (the cis plasmid) and a plasmid containing rep and cap (the trans plasmid), and infecting the cells with a helper virus (e.g., adenovirus or herpes virus).
  • a helper virus e.g., adenovirus or herpes virus.
  • rcAAV pseudo-wildtype replication-competent AAV
  • a second method that has been used to produce rAAV involves co-
  • the trans 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
  • packaging cell lines do not produce high levels of rAAV.
  • 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
  • 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 the native AAV p5
  • promoter upstream o ⁇ rep is removed and replaced with a minimal promoter or with
  • This novel vector when infected into cells containing a nucleic acid sequence comprising a transgene flanked by AAV ITRs, results in the production of high levels of rAAV.
  • the nucleic acid sequence comprising the transgene flanked by 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 carrying the transgene flanked by ITRs (see, e.g, U.S. Pat. No. 5,856,152), infection with an rAAV comprising the transgene, or any other method known in the art, such as transfection, lipofection or microinjection, of plasmid DNA comprising the transgene flanked by ITRs.
  • rep operably linked to a minimal promoter or to no promoter, is inserted into either the El or E3 regions of an adenovirus.
  • the adenovirus is deleted in El or 3 alone, or a combination of both.
  • the adenovirus vector is further deleted in E4.
  • rep sequences may be inserted in E4, while upstream of these rep sequences there may be no promoter or a minimal promoter.
  • 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 advantage of this method is that it is easily scaled for industrial production of 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.
  • the 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 sequences.
  • 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 native p5 promoter of rep is removed and replaced with a minimal promoter or with no promoter.
  • 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
  • Ad-HSP-RC Ad-HSP-RC.
  • M 1 kb DNA ladder size marker (Gibco BRL).
  • LacZ co-infection of 293 cells lane 6, sublOOr and Ad-AAV-LacZ stepwise
  • M 1 kb DNA Ladder size marker (Gibco BRL).
  • Fig. 9 A. Ethidium bromide stained agarose gel of Hirt DNA samples
  • Fig. 9B Southern blot analysis of the gel shown in Fig. 9A
  • Lane 1 lacL DNA fragment as a positive
  • M 1 kb DNA Ladder size marker (Gibco BRL)
  • FIG. 10 Western blot analysis of Rep and Cap protein expression in
  • the instant invention relates to a novel adenoviral vector and a method for producing high titer stocks of rAAV using this vector.
  • the adenoviral vector comprises a rep gene in which the native AAV p5 promoter upstream of the rep coding sequences has been deleted or effectively rendered inactive by mutation or partial deletion and replaced by a minimal promoter or no promoter.
  • Rep78 and Rep68 are produced at much lower levels than Rep52 and Rep40 in 293 cells or other El -complementing cell
  • host cells are infected with an adenovirus vector comprising a rep gene that lacks any promoter.
  • Rep78 and Rep68 protein expressed in host cells infected by an adenovirus lacking any promoter upstream of rep coding sequences are unknown, it is expected that Rep78 and
  • 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.
  • the total amount of Rep78 and Rep68 protein is less than 80%, more preferably less than 50%, of the total amount of Rep52 and Rep40 produced in the infected cells. In a more preferred embodiment, the total amount of Rep78 and Rep68 is less than 25% of the total amount
  • the total amount of Rep78 and Rep68 is less than 15%, more preferably 10%, and more preferably is less than 5% of the total amount of Rep 52 and Rep40 produced in the infected cells.
  • the instant invention demonstrates that adenoviral vectors comprising a rep gene with its native p5 promoter are unstable when infected into host cells while 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 and 3B.
  • Example 4 demonstrates that Ad-p5-RC, which is an adenovirus containing p5, rep and cap in the El site of the adenovirus vector, undergoes a rearrangement or deletion event in the rep-cap DNA sequences of the adenovirus vector when passaged in 293 cells.
  • Ad-HSP-RC which contains a minimal heat shock protein promoter (HSP), rep and cap
  • HSP heat shock protein promoter
  • a minimal promoter or promoterless rep-containing adenovirus of the instant invention is one which is stable upon propagation in a defined host cell system, such as 293 cells.
  • 293 cells express Rep and Cap proteins when co-infected with Ad-HSP- RC and Ad-AAV-LacZ (see lane 5 of Fig. 10), but do not express these proteins when co-infected with Ad-p5-RC and Ad-AAV-LacZ (see lane 4 of Fig. 10).
  • Ad-p5-RC The effect of the deletion or rearrangement in Ad-p5-RC is shown by the levels of rAAV produced using this adenovirus vector. Little or no replicating rAAV is produced in cells co-infected with Ad-p5-RC and Ad-AAVLacZ, 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 amounts of replicating rAAV is produced in cells that have been infected with an adenovirus vector comprising rep sequences downstream of no promoter.
  • a "recombinant adeno-associated virus (rAAV) genome” comprises all or a part of an AAV genome, wherein the viral genome may be wild type or may contain point mutations or deletions, and optionally comprises a transgene operably linked to expression control sequences.
  • the transgene may be regulated in cis or in trans.
  • the rAAV genome comprises a transgene flanked by AAV inverted terminal repeats (ITRs).
  • ITRs AAV inverted terminal repeats
  • the rAAV genome of the invention may be embedded in the genome of an adenovirus vector to form a hybrid Ad/AAV. See U.S. Pat. No. 5,586,152, herein incorporated by reference.
  • 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
  • the 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, termination, promoter and enhancer sequences; efficient RNA processing signals
  • sequences that enhance protein stabihty are sequences that enhance protein stabihty; and when desired, sequences
  • 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
  • adenoviruses that comprise an rAAV genome.
  • adenovirus vector is a recombinant adenovirus comprising one or more foreign genes, wherein the 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 are from any AAV serotype, including AAV Serotypes 1 to 6, as well as any AAV that
  • 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 activity For instance, one may move the p5 promoter
  • a p5 promoter is effectively deleted
  • p5 promoter is effectively deleted when it promotes less than 25% of wildtype p5 promoter activity.
  • 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 is effectively deleted when a host cell infected with an adenovirus comprising rep and 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; more preferably at least 10 4 particles per cell; and, even more preferably, at least 10 5 or 10 6 particles per cell. In general, there are approximately 1 x 10 3 to 3 x 10 3 particles per transducing units (TU). The number of particles required to produce one TU varies based upon the transgene, purification method and assay method.
  • a "minimal promoter” is one that essentially comprises only a TATA box and promotes only very low or basal levels of transcription ofrep7S and rep68.
  • a promoter is a nucleotide sequence that promotes the initiation of transcription at a particular site by the cell's transcriptional machinery.
  • a minimal promoter promotes transcription that is less than 80% of the wildtype p5 promoter, more preferably less than 50% of the wildtype p5 promoter, even more preferably less than 25% of the wildtype p5 promoter. In a more preferred embodiment, a minimal promoter is one that promotes transcription that is less than 20% of the wildtype p5 promoter, even more preferably less than 15% of the wildtype p5 promoter. In an even more preferred embodiment, a minimal promoter is one that promotes transcription that is less than 10% of the wildtype p5 promoter, even more preferably less than 5%, even more preferably less than 1% of the wildtype p5 promoter.
  • a minimal promoter also may be defined by functional measures.
  • a minimal promoter is one in which the rep gene to which it is operably linked is Hot rearranged or deleted when an adenovirus comprising the minimal promoter and rep gene is infected into a host cell, such as 293 cells.
  • a minimal promoter is one in which a host cell infected with an adenovirus comprising a minimal promoter that
  • the titer is at least 10 2 particles per cell; preferably at least 10 3 particles per cell; more preferably at least 10 4 particles per cell; and, even more preferably, at least 10 s or 10 6 particles per cell.
  • an artificial minimal promoter may be constructed by using a sequence or a consensus sequence of a TATA box and adding nucleotide sequences to the 5' and 3' ends of the TATA box.
  • the activity of the minimal promoter may be measured by measuring the transcription of the artificial minimal promoter and comparing it to an natural minimal 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 has been inserted in its place.
  • 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 a promoter that was active in bacterial or insect cells, for example, but that was inactive in a mammalian host cell.
  • rep78/68 is promoterless when the p5 promoter has been deleted and replaced by an inducible promoter that permits low-level expression of rep78/68.
  • adenoviruses and adenoviral vectors are known, including human adenoviruses types 1-46, chimpanzee adenoviruses, canine adenoviruses, bovine adenoviruses [all available from the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209], and ovine adenoviruses (Both et al., WO 97/06826 Al). Any of these adenoviruses may be used in this invention, provided that the adenovirus is able to infect the target host cell.
  • the adenoviral vector comprises the AAV rep gene downstream of a minimal promoter or no promoter (this is alternatively referred to as a minimal promoter or promoterless AAV rep gene), and sufficient helper virus functions for rAAV production in a host cell.
  • the adenoviral vector further comprises the AAV cap gene.
  • adenoviral sequences required for replication and encapsidation of the rAAV genome depends upon whether the host cell expresses any helper functions or whether other vectors or viruses are introduced into the host cell which express helper functions. For instance, if the adenovirus is to be used to infect a cell line that expresses El, e.g., the 293 cell line, then the adenoviral vector could comprise rep and cap, and could also comprise those helper virus functions required in addition to El (e.g., E2a, E4ORF6 and VAI RNA).
  • the adenovirus is used to infect a cell line such as 84-31, which expresses El and E4, then the adenoviral 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, may comprise E4ORF6 and VAI RNA. In an alternative embodiment, helper functions may be supplied by chemical or physical methods or by other helper viruses.
  • 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 produced using homologous recombination.
  • the recombinant adenovirus is produced using Cre-/ ⁇ x recombination (12).
  • helper virus functions may be provided by nucleic acid sequences that are introduced into the host cell.
  • 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 and further comprises helper functions that are not expressed by either the host cell or the adenovirus comprising the rep gene. See, e.g., Examples 6-8.
  • some or all of the helper virus functions may be provided by any method known in the art, such as by transfection or direct injection, as discussed above. Based on this description, other embodiments of the adenoviral vector will be readily apparent to those of ordinary skill in the art.
  • Other viral vectors in which Rep interferes with viral replication also may be used. Rep and Cap Nucleic Acids
  • the Rep and Cap proteins may have a naturally
  • the rep and cap genes are derived from the same
  • the rep and cap genes are independently selected from the rep and cap genes.
  • 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
  • the Rep and/or Cap proteins may have
  • a mutated sequence including insertions, deletions, fragments or point mutations of
  • 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.
  • adenoviral vector e.g., WO 98/27204.
  • 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
  • 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
  • a TATA box and an Spl box corresponds to nucleotides 3511 to
  • 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.
  • 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 (HS V-2), cytomegalovirus (CMV) and pseudorabies virus (PRV)] or by exposure of the cells to different chemical or physical agents.
  • HSV-1 herpes simplex virus type 1
  • HS V-2 type 2
  • CMV cytomegalovirus
  • PRV pseudorabies virus
  • helper functions are required for high levels of rAAV production.
  • the rAAV genome may be stably integrated into the host cell or may be transfected or infected into the host cell by methods known in the art.
  • After transfecting the host cell with the nucleic acid encoding the potential helper function one may then measure the titer of the rAAV that is produced to determine if the nucleic acid encodes a helper function.
  • the helper functions are nucleic acids derived from a virus.
  • the helper functions are derived from adenovirus types 2 or 5, HSV-1, HSV-2, CMV or PRV.
  • the helper functions are Ela, Elb, E2a, E4ORF6 proteins and VAI RNA from adenovirus.
  • the nucleic acid encodes the helper functions from the helicase-primase complex of HSV (UL5, UL8 and UL52) and the major single-stranded DNA binding protein of HSV (UL29).
  • helper functions for recombinant AAV may be provided by chemical or physical agents, including ultraviolet light, cycloheximide, hydroxyurea and various carcinogens.
  • the required helper functions for production of a rAAV may be delivered to the host cell by any method known in art.
  • the helper functions may be delivered by transfection with a vector, such as a plasmid, by infection with a viral vector comprising the helper functions, or by any other method known in the art, including those discussed above (e.g., biolistic injection of DNA, use of DNA conjugates, etc.).
  • 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.
  • some of the helper functions may be expressed by the mammalian cell line while other helper functions are introduced by a vector.
  • the helper functions are transduced into the host cells by an adenovirus. In a more preferred embodiment, some or all of the helper functions are transduced into the host cell by the adenovirus that comprises the rep and/or cap genes.
  • the native helper function sequences are used. However, mutated helper function sequences may be used so long as they retain their helper function activity.
  • the helper function nucleic acids 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 promoter/enhancer or the zinc-inducible metallothionein promoter, respectively, as known in the art or as described above.
  • the rAAV Transgene Cassette 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 promoter/enhancer or the zinc-inducible metallothionein promote
  • transgene begins with a desired transgene, then associates the transgene with appropriate expression regulatory sequences (ERS), e.g., promoter, enhancer, polyadenylation site, then inserts this ERS-transgene construct between AAV flanking sequences, e.g., the ITRs, in place of rep and cap genes normally found therein.
  • ERS expression regulatory sequences
  • AAV flanking sequences e.g., the ITRs
  • the transgene cassette comprised of the ERS-transgene bordered by the AAV flanking sequences may then be embedded in an adenovirus vector separate from that which carries the rep gene.
  • the transgene cassette may be inserted into a plasmid vector and
  • 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 be introduced transiently, such as by infection with a hybrid Ad/ AAV virus. See, e.g., Examples 3 and 6-8. Each element of the transgene cassette is further described below:
  • the Transgene 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 antibiotics, or a reporter gene allowing detection, e.g., by color in the case of the use of green fluorescent protein.
  • the transgene may be one that is
  • a transgene may be a normal gene
  • transgene that replaces or augments the function of a patient's defective gene.
  • 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
  • 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
  • genes which are normally expressed in cells but whose products are produced in abnormal amounts due to over- or under-expression are normally expressed in cells but whose products are produced in abnormal amounts due to over- or under-expression.
  • the appropriate gene for expression is one which expresses a normal gene product
  • Transgenes used for production of proteins in vitro include proteins such as secreted factors, including hormones, growth factors
  • composition of the transgene sequence depends upon the intended use for the resulting rAAV.
  • one type of transgene sequence comprises a reporter or marker sequence, which upon expression produces a detectable signal.
  • reporter or marker sequences include, without limitation, DNA sequences encoding E.
  • coli ⁇ -lactamase ⁇ -galactosidase (LacZ)
  • alkaline phosphatase HSV thymidine kinase
  • green fluorescent protein GFP
  • CAT bacterial chloramphenicol acetyltransferase
  • firefly luciferase eukaryotic membrane bound proteins including, for example, CD2, CD4, CD8, the influenza hemagglutinin protein, and others well known in the art, to which high affinity antibodies directed to them exist or can be made routinely, and fusion proteins comprising a membrane bound protein appropriately fused to an antigen tag domain from, among others, hemagglutinin or myc.
  • sequences when associated with regulatory elements which drive their expression, provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectroscopic assays, fluorescent activated cell sorting assay and immunological assays, including ELISA, RIA and immunohistochemistry.
  • the transgene is the LacZ gene
  • the presence of a rAAV is detected by assays for ⁇ -galactosidase activity.
  • 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 proteins, sense or antisense nucleic acids (e.g., RNAs), or
  • catalytic RNAs The invention may be used to correct or ameliorate gene
  • 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 are able to bypass
  • polio elements from hepatitis C and members of the picornavirus family (e.g., polio and
  • IRES elements can be linked to heterologous proteins
  • each open reading frame is
  • the insert size can be no greater than approximately 4.8 kilobases; however, for an adenovirus which lacks all of its helper functions, the insert size can be no greater than approximately 4.8 kilobases; however, for an adenovirus which lacks all of its helper functions, the insert size can be no greater than approximately 4.8 kilobases; however, for an adenovirus which lacks all of its helper functions, the insert size can be no greater than approximately 4.8 kilobases; however, for an adenovirus which lacks all of its helper functions, 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 factor (GRF), thyroid stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), prolactin, melatonin, vasopressin, ⁇ -endorphin, met-enkephalin, leu- enkephalin, prolactin-releasing factor, prolactin-inhibiting factor, corticotropin- releasing hormone, thyrotropin-releasing hormone (TRH), follicle stimulating hormone (FSH), luteinizing hormone (LH), chorionic gonadotropin (CG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, endostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), bF
  • cytokines and lymphokines such as thrombopoietin (TPO), interleukins (IL) IL-l ⁇ , IL-l ⁇ , IL-2, B -3, IL-4, B -5, IL-6, E -7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, B -16, and IL-17,
  • 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.
  • 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 trie 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-2, ICAM-3 and ICAM-4
  • VCAM vascular cell adhesion molecules
  • 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 jun,fos, max, mad, serum response factor (SRF), AP-1, AP-2, myb, MRG1,
  • IRF-1 interferon regulation factor 1
  • ETS-binding protein STAT
  • GAT A-box binding proteins e.g., GATA-3
  • transcarbamylase arginosuccinate synthetase, arginosuccinate lyase, arginase,
  • fumarylacetoacetate hydrolase phenylalanine hydroxylase, alpha- 1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor VH, factor VIII, factor IX, factor II, factor V, factor X, factor XII, factor XI, von Willebrand factor,
  • angiotensin converting enzyme endothelin-1, atrial natriuetic peptide, pro- urokinase, urokinase, plasminogen activator, heparin cofactor II, activated protein C (Factor V Leiden), Protein C, antithrombin, cystathione beta-synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl 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- protein, T-protein, Menkes disease protein, tumor suppressors (e.g., p53), cystic fibrosis transme
  • PWD Cu/Zn superoxide dismutase, aromatic aminoacid decarboxylase, tyrosine hydroxylase, acetylcholine synthetase, prohormone convertases, protease inhibitors, lactase, lipase, trypsin, gastrointestinal enzymes including chyromotrypsin, and pepsin, adenosine deaminase, ⁇ l anti-trypsin, tissue inhibitor of metalloproteinases (TIMP), GLUT-1, GLUT-2, trehalose phosphate synthase, hexokinases I, II and III, glucokinase, any one or more of the individual chains or types of collagen, elastin, fibronectin, thrombospondin, vitronectin and tenascin, and suicide genes such as thymidine kinase and cytosine deaminase.
  • TMP tissue inhibitor of
  • proteins include those involved in lysosomal storage disorders, including acid ⁇ -glucosidase, ⁇ - 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 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
  • 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; herpes simplex viruses types 1 and 2; varicella-zoster virus; human herpes virus type 6, 7
  • transgenes may also be directed against peptides from tumor antigens to provide immunization for tumors and cancers.
  • a great number of expression control sequences ⁇ native, constitutive, inducible and/or tissue-specific — are known in the art and may be utilized to drive expression of the transgene and the nucleic acid sequences
  • expression control sequences typically include a promoter, an enhancer, such as one derived from an immunoglobulin gene, SV40, cytomegalovirus, etc., and a polyadenylation sequence.
  • the polyadenylation sequence generally is inserted following the transgene sequences and before the 3' flanking sequence of the transgene.
  • a transgene-carrying molecule useful in the present invention may also contain an intron, desirably located between the promoter/enhancer sequence and the transgene.
  • One possible intron sequence is also derived from SV40 and is referred
  • IRES internal ribosome entry site
  • An IRES element can be used for the transgene or for any of the other nucleic acid sequences encoding the replication and encapsidation polypeptides, the helper functions or the ligand. Selection of these and other common vector elements are conventional and many such sequences are available [see, e.g., Sambrook et al, and references cited therein at, for example, pages 3.18-3.26 and 16.17-16.27 and Ausubel et al., Current Protocols in Molecular Biology. ohn Wiley & Sons, New York, 1989]. In one embodiment, high-level constitutive expression will be desired.
  • promoters examples include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter/enhancer, the cytomegalovirus (CMV) immediate early promoter/enhancer [see, e.g., Boshart et al, Cell. 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic ⁇ -actin promoter and the phosphoglycerol kinase (PGK) promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • inducible promoters may be desired.
  • Inducible promoters are those which are regulated by exogenously supplied compounds, either in cis or in trans, including without limitation, the zinc-inducible metallothionine (MT) promoter; the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system [WO 98/10088]; the ecdysone insect promoter [No et al, Proc. Natl. Acad. Sci. USA. 93:3346-3351 (1996)]; the tetracycline-repressible system [Gossen et al, Proc. Natl. Acad. Sci. USA. 89:5547-5551 (1992)]; the tetracycline-inducible
  • promoters which may be useful in this context are those which are regulated by a
  • nucleic acid sequence of interest will be used.
  • the native promoter may be any promoter
  • the native promoter may be used
  • 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
  • a promoter active in muscle may be used. These include the promoters from genes encoding skeletal ⁇ -actin, myosin light chain 2A,
  • dystrophin muscle creatine kinase
  • synthetic muscle promoters with activities higher than naturally-occurring promoters [see Li et al., Nat. Biotech..
  • 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..
  • NSE neuron-specific enolase
  • host cell of choice may be selected by one of skill in the art using the guidance
  • transgene by selecting one or more expression control sequences and operably linking the expression control sequence to the nucleic acid sequence to be regulated. Then, one may insert these operably linked sequences comprising the
  • 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,
  • AAV ITRs consist of repeated sequences
  • the AAV ITRs are required for replication, excision and
  • 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.
  • a host cell used in this invention is one that may be infected by the adenovirus vector of the instant invention.
  • Another preferred characteristic of the host cell is that it is able to replicate the rAAV at high levels.
  • Appropriate host cells include, without limitation, CHO, BHK, MDCK and various murine cells, e.g., 10T1/2 and WEFH 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), human embryonic kidney (HEK), human embryonic lung (HEL) and HT1080 cells.
  • CHO, BHK, MDCK and various murine cells e.g., 10T1/2 and WEFH cells
  • African green monkey cells such as VERO, COS 1, COS 7, BSC 1, BSC 40, and BMT 10
  • human cells such as WI38, MRC5, A549, human embryonic retinoblast (HER), human embryonic kidney (HEK), human embryonic lung (HEL) and HT1080 cells.
  • appropriate cells include 293 cells (human embryonic kidney cells that express adenoviral El a and Elb proteins), 911 or PER.C6 cells (human embryonic retinoblast cells that express adenoviral El; see WO 97/00326), B50 cells (HeLa cells that express AAV rep and cap, see PCT US98/19463), 84-31 cells (293-based cells that express adenovirus Ela, Elb and E4, Ref. 4), 10-3 cells (293-based cells that express adenovirus Ela, Elb and E4ORF6, Ref.
  • 293 cells human embryonic kidney cells that express adenoviral El a and Elb proteins
  • 911 or PER.C6 cells human embryonic retinoblast cells that express adenoviral El; see WO 97/00326)
  • B50 cells HeLa cells that express AAV rep and cap, see PCT US98/19463
  • 84-31 cells (293-based cells that express adenovirus Ela,
  • 3T3 cells mouse embryonic fibroblast cell line
  • NIH3T3 cells subline of 3T3 cells
  • HepG2 cells human liver carcinoma cell line
  • Saos-2 cells human osteogenic sarcoma cell line
  • HuH7 cells or HeLa cells human carcinoma cell line
  • 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
  • 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
  • the 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
  • 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 promoter and cap, and the other adenovirus comprising the rAAV genome. Infection of host cells by adenovirus is highly efficient and may be easily scaled to a large number of cells.
  • the instantly described method produces rAAV at a high titer.
  • the titer is at least 10 2 particles per cell; preferably at least 10 3 particles per cell; more preferably at least 10 4 particles per cell; and, even more preferably, at least 10 5 or 10 6 particles per cell.
  • the instant invention also encompasses lysates and supematants of host cells comprising rAAV. These lysates and supematants differ from those produced by prior art methods because of the higher level of rAAV contained therein without concentration.
  • the rAAV produced by the method of this invention may be formulated as a pharmaceutical or pharmacological composition for use for any form of transient and stable gene transfer in vivo and in vitro.
  • the composition comprises at least the rAAV and a pharmaceutically acceptable carrier.
  • the rAAV may be used for in vivo and ex vivo gene therapy, genetic immunization, in vitro protein production and diagnostic assays.
  • the rAAV may be introduced into cells ex vivo or in vivo. Where the virus is introduced into a cell ex vivo, 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
  • the rAAV is administered in sufficient amounts to infect the desired
  • transgene or viral gene products in the case of a vaccine
  • rAAV intramuscular; subcutaneous; intradermal; oral and other parenteral routes of administration. Routes of administration may be combined, if desired. Dosages of rAAV will depend primarily on factors such as the
  • an effective human dosage of rAAV is generally in the range of from about 0.5 ml to 50 ml of saline solution containing rAAV at concentrations of 1 x 10 7 or 1 x 10 8 or 1 x 10 9 or 1 x 10 10 or 1 x 10 u or 1 x 10 12 or 1 x 10 13 or 1 x 10 14 or 1 x 10 15 or 1 x 10 16 particles
  • the dosage will be adjusted to balance the corrective benefits against any adverse side effects.
  • the levels of expression of the selected gene may be monitored to determine the type and frequency of dosage administration.
  • 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- derived 84-31 cells (1) which express adenovirus El/E4orf6 proteins, and HeLa- derived B50 cells (7) which express AAV-2 Rep and Cap proteins from the native p5 promoter, are obtained from Dr. Guangping Gao, Institute for Human Gene Therapy, University of Pennsylvania.
  • 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).
  • Human adenovirus type 5 (ATCC VR-5) and derived recombinant adenoviruses are propagated on 293 cells and purified through CsCl gradient centrifugation accordmg to the method of Jones and Shenk with
  • DNA containing the rep and cap sequences of pAV2 (ATCC 37216) between Drain site (nucleotide 241, upstream of the AAV-2 p5 promoter) and Ncol site (nucleotide 4489, downstream of the polyA signal) is removed and replaced through multiple cloning steps with a DNA cassette containing GFP under the transcriptional control of elongation factor 1 alpha
  • Fig.1 The AAV-2 rep and cap genes located between a Dra III site (nucleotide 241, upstream of the p5 promoter) and a Bsal site (nucleotide 4464, downstream of the polyA signal) are further subcloned to obtain pAd-p5-RC (Fig.1).
  • pAd-p5-RC Fig.1
  • a small DNA fragment between nucleotides 241 and 287 of pAd-p5-RC containing the p5 promoter is removed and replaced with a Drosophila melanogaster minimal heat shock protein (HSP) promoter from pIND (Invitrogen) to create pAd-HSP-RC (Fig.l).
  • HSP Drosophila melanogaster minimal heat shock protein
  • Recombinant adenoviruses Ad-p5-RC and Ad-HSP-RC 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 production of rAAV.
  • pAV2cisEFGFP by the calcium phosphate transfection method.
  • the monolayer is replenished with fresh medium containing 10% FBS and cultured for 24 hours. Following trypsinization, cells are seeded at a 1 :20 dilution in fresh medium containing 10% FBS. After incubation for another 24 hours, fresh medium
  • G418-resistant cells The medium containing G418 is replaced every 3-4 days to allow formation of G418-resistant cell colonies. A total of fifty colonies are picked, six of which demonstrate constitutive GFP expression. These six clones are expanded and tested for their ability to rescue functional rAAV by transfection with pBV-EiOV-RC, a plasmid that carries adenovirus E2A, E4ORF6, and VAI genes as well as AAV rep-cap genes.
  • One cell clone, 293-CG3 shows high efficiency of rAAV rescue and is expanded and used for further experiments.
  • PCR polymerase chain reaction
  • HC#30 (5'-CGTAACCGAGTAAGATTTGG-3'; SEQ ID NO: 1)
  • HC#31 (5'-ATGTTGGTGTTGGAGGTGAC-3'; SEQ ID NO: 2)
  • HC#33 (5'-AGCCTTGACTGCGTGGTGGT-3'; SEQ ID NO: 4),
  • HC#34 (5'-GTACCTGTATTACTTGAGCA-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'-GACCCAGACTACGCTGACGA-3'; SEQ ID NO: 8).
  • spermine-HCl and supernatant is collected. The supernatant is then treated with
  • Adenoviral DNA in the supernatant is precipitated with isopropanol and dissolved in TE RNase buffer (10 mM Tris-HCl, pH8.0, 1 mMEDTA, 20 ⁇ g/ml RNase).
  • PCR assay is performed using the Robocycler Gradient 96 thermal cycler (Stratagene), PCR products are separated on a 1% agarose gel and DNA is stained with ethidium bromide. As shown in Fig. 3, all four expected DNA PCR products are obtained when using Ad-HSP-RC DNA as template, indicating
  • Ad-HSP-RC Production of rAAV through Infection of 293-CG3 Cells with Ad-HSP-RC Since Ad-HSP-RC is shown to contain full length AAV-2 rep-cap
  • DNA sequences its utility to produce rAAV is analyzed.
  • 293-CG3 cells are seeded in 6-well plates at a density of 1.0x10 6 cells/well. Twelve to fifteen hours later, the culture media is removed from the cell monolayer and dilutions of Ad-HSP-RC virus in 0.5 ml of serum-free DMEM are added to the cells. Following a 30 min. incubation, an additional 2.5 ml of DMEM containing 10% FBS is added and the infection is allowed to proceed for a total of 3 days. Infected cells are harvested and pelleted.
  • lysis buffer 50 mM Tris-HCl, pH7.4, 1.0 mM MgCl 2 , 0.5% DOC
  • sonication for 3x1 min.
  • Cell debris is removed by centrifugation at 3,000 rpm using a Beckman GS-6R centrifuge for 10 min. The lysate supernatant is collected for titration of rAAV.
  • 84-31 cells (1) are plated 3-4 hours before use on 24-well plates at a density of 2x10 s cells/well.
  • lysate is diluted in DMEM containing 10% FBS and heated at 56° C for 60 min to
  • rAAV lysate is added to the monolayer, cells are incubated for approximately 24 hours, and GFP-expressing cells are scored as transducing units (TU).
  • the results demonstrate that rAAV is successfully 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- HSP-RC. Using an MOI of 250 particles/cell, as much as 70 TU/cell of rAAV is produced. However, further increasing the MOI of the Ad-HSP-RC virus does not have compared to produce a multiplicity of infection (MOI) of Ad- HSP-RC.
  • MOI multiplicity of infection
  • AAV vector sequences are stably integrated into the host cell chromosome while rep-cap functions necessary for their rescue and packaging into rAAV are provided by Ad-HSP-RC.
  • Ad-HSP-RC Rep-cap functions necessary for their rescue and packaging into rAAV
  • Ad-HSP-RC Ad-HSP-RC.
  • 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
  • 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
  • experiment carries lacZ as a transgene. Briefly, 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
  • LacZ can produce high titers of rAAV in 293 cells.
  • the data indicate that with
  • AAV-LacZ does not increase the rAAV yield. While the conventional method for
  • rAAV production using plasmid co-transfection is limited in its yield of rAAV, the
  • Infected cells are harvested and
  • Ad-HSP-RC and Ad-AAV-LacZ 100 particles/cell each of Ad-HSP-RC and Ad-AAV-LacZ are used to infect 293 cells for various times.
  • AAV-LacZ hybrid genome extrachromosomal DNA is analyzed in 293 cells co-
  • the control system is based on the use of the B50 cell line, that was previously
  • B50 cells are initially infected with sublOOr, an adenovirus temperature-sensitive mutant in the E2b gene, to induce rep and cap expression and
  • adenovirus in which the AAV vector sequence is cloned in the El region of a
  • the rAAV genome delivered by the hybrid vector is rescued, replicated and
  • 293 cells are grown in 10-cm dishes to subconfluency and are either
  • Ad-HSP-RC co-infected with Ad-HSP-RC plus Ad-AAV-LacZ or with Ad-p5-RC plus Ad-
  • AAV-LacZ at 200 particles/cell of each virus.
  • Negative controls include single virus infections of 293 cells with either Ad-p5-RC,
  • Ad-HSP-RC or Ad-AAV-LacZ alone are harvested 72 hours post-replasia
  • Hirt solution 10 mM Tris-HCl, pH 7.4, 100 mM
  • EDTA 0.6% SDS
  • the lysate is mixed with 0.25 ml of 5 MNaCl and incubated at
  • Ad-HSP-RC (Fig. 8, lane 2)
  • Ad-AAV-LacZ (Fig. 8, lane 3).
  • Ad-p5-RC and Ad-AAV-LacZ also does not result in the formation of either rAAV DNA species (Fig. 8, lane 5), suggesting that Ad-p5-RC
  • the Rep proteins are detected using monoclonal antibody
  • AAV Rep proteins may interfere with adenovirus replication

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)

Priority Applications (6)

Application Number Priority Date Filing Date Title
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
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
US10/089,394 US7115391B1 (en) 1999-10-01 2000-09-29 Production of recombinant AAV using adenovirus comprising AAV rep/cap genes
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
US11/509,855 US20070065412A1 (en) 1999-10-01 2006-08-25 Production of recombinant AAV using adenovirus comprising AAV rep/cap genes

Applications Claiming Priority (2)

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US15746699P 1999-10-01 1999-10-01
US60/157,466 1999-10-01

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DK2359869T3 (en) * 2001-12-17 2019-04-15 Univ Pennsylvania Sequences of adeno-associated virus (AAV) serotype 8, vectors containing these, and uses thereof
WO2008156681A2 (fr) * 2007-06-14 2008-12-24 Yoshihiro Kawaoka Vecteurs d'adénovirus pour la production de virus de la grippe
PL2947149T3 (pl) * 2007-06-21 2018-09-28 Alphavax, Inc. Kasety bez promotora do ekspresji alfawirusowych białek strukturalnych
DK2368903T3 (da) * 2010-03-26 2014-05-05 Deutsches Krebsforsch Adenovirusafledt hjælpevirus til fremme af produktion af rekombinant parvovirus
MX2016000345A (es) 2013-07-12 2016-06-21 Philadelphia Children Hospital Vector aav y ensayo para anticuerpos neutralizantes anti-aav (virus adenoasociado).
LT3116900T (lt) 2014-03-09 2020-12-10 The Trustees Of The University Of Pennsylvania Kompozicijos, naudotinos ornitino transkarbamilazės (otc) nepakankamumo gydymui
US10053671B2 (en) 2014-06-20 2018-08-21 Wisconsin Alumni Research Foundation (Warf) Mutations that confer genetic stability to additional genes in influenza viruses
ES2751919T5 (es) * 2014-11-28 2023-10-04 Uniqure Ip Bv Impurezas de ADN en una composición que comprende un virión parvoviral
EA201791630A1 (ru) * 2015-01-20 2018-04-30 Джензим Корпорейшн Аналитическое ультрацентрифугирование для определения характеристик рекомбинантных вирусных частиц
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US5856152A (en) * 1994-10-28 1999-01-05 The Trustees Of The University Of Pennsylvania Hybrid adenovirus-AAV vector and methods of use therefor
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