EP1180158A1 - Compositions and methods for production of recombinant virus using a carrier vector derived from a nonmammalian virus - Google Patents
Compositions and methods for production of recombinant virus using a carrier vector derived from a nonmammalian virusInfo
- Publication number
- EP1180158A1 EP1180158A1 EP00936317A EP00936317A EP1180158A1 EP 1180158 A1 EP1180158 A1 EP 1180158A1 EP 00936317 A EP00936317 A EP 00936317A EP 00936317 A EP00936317 A EP 00936317A EP 1180158 A1 EP1180158 A1 EP 1180158A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- virus
- recombinant
- carrier
- replication
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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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
- C12N2710/14011—Baculoviridae
- C12N2710/14041—Use of virus, viral particle or viral elements as a vector
- C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
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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
- C12N2710/14011—Baculoviridae
- C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
- C12N2710/14141—Use of virus, viral particle or viral elements as a vector
- C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N2710/24011—Poxviridae
- C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
- C12N2710/24141—Use of virus, viral particle or viral elements as a vector
- C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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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
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/108—Plasmid DNA episomal vectors
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- C12N2830/00—Vector systems having a special element relevant for transcription
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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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- 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
- C12N2830/60—Vector systems having a special element relevant for transcription from viruses
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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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
- C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
Definitions
- This invention relates to novel nonmammalian carrier vectors and 5 viruses useful in the production of high titers of recombinant viruses which may
- the nonmammalian carrier vector (“carrier
- vector is a chimeric vector which includes those portions of a nonmammalian
- 0 vector includes various nucleic acid cassettes, which may include an embedded
- the invention also provides methods of producing high concentrations of recombinant
- 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 which 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.
- 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 that replaces or augments the function of a patient's defective gene
- the transgene may be a normal gene 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
- a transgene may also be used for immunization against various conditions
- the transgene also may be one which is useful for production of proteins in vitro, such as for large-scale production of therapeutic
- RNAs expressed from mutated genes or modifies or destroys viral RNAs
- Transgenes used for production of proteins in vitro include proteins such as secreted factors, including hormones, growth factors and enzymes.
- 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.
- disorders such as anemia, in which there is insufficient production of red blood
- EPO erythropoietin
- 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
- Certain methods are amenable to targeted delivery of the exogenous gene to specific tissues, such as liver tissue.
- One method of delivering genes to specific cells relies upon the function of a cell-specific receptor.
- ASGP-R asialoglycoprotein receptor
- hepatocytes (Spiess et al., 1990, Biochem. 29: 10009-10018), is a lectin which has
- the recombinant virus containing a transgene is
- type or modified virus may result in recombinant virus stocks contaminated with wild-type or modified virus Supplying plasmids encoding the required gene
- viruses include retrovirus, hepatitis B virus (HBV),
- AAV adeno-associated virus
- herpesvirus adenovirus, adeno-associated virus (AAV) and herpesvirus.
- AAV possesses unique
- AAV is a parvovirus, the genome of which is about 4.7 kb in length
- the AAV genome including 145 nucleotide inverted terminal repeats (ITRs).
- ITRs 145 nucleotide inverted terminal repeats
- Rep polypeptides (repl , rep68, rep52, and rep40) are involved in
- Cap proteins (VP1, VP2,
- the 5' and 3' ends of the AAV genome are the 145 bp ITRs, the first 125 bp of
- the ITRs represent the minimal sequence required for replication, rescue,
- a cell is coinfected with a lytic helper virus, such as adenovirus (Ad) or herpesvirus.
- a lytic helper virus such as adenovirus (Ad) or herpesvirus.
- the AAV provirus Upon infection with a helper virus, the AAV provirus is rescued and amplified, and
- rescued AAV genomes are packaged into preformed protein capsids (icosahedral symmetry approximately 20 nm in diameter) and released as infectious virions that
- a recombinant virus capable of delivering the transgene to target host cells.
- helper virus or additional plasmids. Furthermore, the requirement by AAV for helper virus
- helper viruses either wildtype or crippled viruses
- (rAAV) vectors comprises co-transfecting eukaryotic cells with a plasmid
- helper virus e.g., adenovirus or herpes
- helper virus with infection by a helper virus is inefficient and cannot be easily scaled up for industrial production of rAAV.
- a second method that has been used to produced rAAV involves a triple plasmid transfection of eukaryotic cells.
- this method one plasmid carries
- transgene and ITRs encodes the rep and cap
- the trans plasmid genes (the trans plasmid), and the third plasmid encodes the helper virus functions, i e adenoviral genes such as El a, El b, E2a and E4 (the helper plasmid)
- the helper virus functions i e adenoviral genes such as El a, El b, 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 (Ad) helper See U S Pat No 5,658,785 Alternatively, the packaging cell line
- a hybrid Ad/ AAV in which a hybrid Ad vector caries the cis
- Nonmammalian viruses have been used to transiently express particular individual exogenous proteins in either mammalian or non-mammalian cells
- viruses of the family Baculoviridae, or "baculoviruses" which are viruses of the family Baculoviridae, or "baculoviruses", which are viruses of the family Baculoviridae, or "baculoviruses", which are viruses of the family Baculoviridae, or "baculoviruses", which are viruses of the family Baculoviridae, or "baculoviruses", which are viruses of the family Baculoviridae, or "baculoviruses", which
- Baculoviruses have also been reported to enter mammalian cells, and baculoviral DNA has been detected in nuclear extracts of
- reporter gene product being carried into the cell after a prolonged incubation of the
- the exogenous gene is not expressed de
- the genome of the baculovirus may be modified by insertion of ligand DNA, which
- the mammalian cells allows only for transient expression of the transgene within the
- AAV recombinant virus for pharmaceutic applications.
- AAV recombinant virus for pharmaceutic applications.
- helper virus which must be inactivated and/or removed from the final products prior to pharmaceutical application.
- the invention exploits the properties of nonmammalian and
- mammalian viruses to create novel chimeric vectors and viruses for the manufacture of an essentially homogeneous recombinant virus preparation in the absence of
- the essentially homogeneous recombinant virus may be used for
- transduction into a cell are required, e.g , in the production of expression libraries
- the carrier vector of the invention is a chimeric vector backbone
- derived from the nucleic acid of a nonmammalian virus includes one or more of
- nucleic acid sequences which encode proteins required for replication and encapsidation of the recombinant virus genome, 3) nucleic acid sequences encoding helper functions (if
- the recombinant virus to be produced is helper-dependent, e.g., AAV), 4) nucleic acid
- the carrier vector may also include any other nucleic acid sequences that are
- one or more carrier vectors may be any suitable carrier vectors.
- recombinant vector produced For instance, if a recombinant AAV vector is desired and the host cell line is one which has rep and cap stably integrated in its genome,
- the carrier vector or vectors would comprise 1) an embedded recombinant viral genome comprising the AAV ITRs and the transgene and 2) separate helper functions, which may include any nucleic acid sequence required for replication and
- helper functions may include any
- recombinant AAV vector is to be produced in a host cell line that does not express
- the carrier vector or vectors may also include the DNA sequences
- the carrier if a recombinant retrovirus is desired, the carrier
- vector or vectors would comprise 1) an embedded recombinant viral genome
- retroviral LTRs or from a heterologous promoter and 2) DNA sequences encoding anv one or a combination o ⁇ gag, pol and env for the functions of replication and
- single carrier vector having all functions not supplied by a host cell increases the efficiency of transduction, and can be more easily scaled for industrial production of the embedded recombinant virus
- the earner vector comprises an
- helper virus or a plasmid
- the embedded recombinant viral genome may comprise a transgene and DNA elements required for replication of a mammalian virus
- the transgene comprises the gene of interest, regulatory elements to regulate its expression, and
- transgene is flanked by the DNA elements required
- retrovirus or the ITRs of adenovirus.
- the recombinant viral genome is embedded
- the recombinant viral genome does not contain a transgene but the recombinant viral genome itself contains point mutations or deletions.
- the point mutations or deletions In this embodiment, the point mutations or
- deletions function to attenuate the replication of the subsequently-produced
- the attenuated recombinant virus may be any virus which could
- picornaviruses such as
- poliovirus hepatitis viruses such as hepatitis B and hepatitis C; cold-adapted
- RSV respiratory syncytial virus
- cold-adapted influenza virus influenza virus
- parainfluenza virus types 1, 2 and 3 parainfluenza virus types 1, 2 and 3
- rotavirus rotavirus
- the carrier vector is replication-proficient in its native host cells.
- a baculovirus backbone results in a chimeric carrier vector
- the embedded recombinant viral genome is unable to excise, replicate, and package into virions because its promoters are inactive in insect cells.
- the carrier vector does not
- recombinant virus are expressed such that the recombinant viral genome is
- the recombinant virus encapsidated, which yields an infectious recombinant virus.
- the recombinant virus encapsidated, which yields an infectious recombinant virus.
- the recombinant virus encapsidated, which yields an infectious recombinant virus.
- the recombinant virus is replication-
- the recombinant virus lacks part or all of the coding regions of
- virus is helper-dependent, such as rAAV, the recombinant virus lacks both
- the recombinant virus in which the recombinant virus is not helper-dependent, the recombinant virus lacks
- recombinant virus may be produced without the need for coinfection
- the invention yields lysates of substantially pure and
- This invention thus has many advantages over current methods for manufacturing recombinant viruses. These advantages include: (l) the
- nonmammalian virus backbone permits insertion of large DNA sequences without
- mammalian cells e.g., AAV rep, VSV-G, retroviral envelope proteins, eukaryotic regulatory proteins, etc.
- nonmammalian viruses do not replicate in mammalian cells, precluding
- the present invention includes nonmammalian
- the nonmammalian carrier in a preferred embodiment, the nonmammalian carrier
- vector contains all the elements required to produce a replication-deficient recombinant viral vector.
- a single viral vector contains all the elements required to produce a replication-deficient recombinant viral vector.
- a single vector contains all the elements required to produce a replication-deficient recombinant viral vector.
- nonmammalian carrier vector contains all the required elements to produce a
- the nonmammalian carrier vector is a baculovirus.
- the invention includes a method of
- the method produces replication-deficient recombinant viral vector lysates and stocks that are free of helper or other contaminating virus.
- the method
- the method is one in which a high titer of
- the invention includes attenuated
- Figure 1 is a schematic diagram of recombinant baculoviruses with
- Figures 2A and 2B represent a genetic map of AAV type 2 Figure
- FIG. 2 A is a schematic representation of the viral genome rep encodes replication
- promoters A, polyadenylation.
- Figure 3 is a schematic diagram of constructed plasmids used in this
- Figure 4 shows the steps involved in rAAV production by traditional
- Figure 5 shows the steps required for rAAV production through the
- BV-EiOV-RC BV-EiOV-RC
- BV-cisEFGFP BV-EiOV-RC
- Figure 6 shows the steps required for rAAV production through the
- Figure 7 shows the steps required for rAAV production through the
- a "recombinant viral genome” comprises all or a part of a viral
- viral genome wherein the viral genome may be wild type or may contain point mutations
- transgene operably linked to expression
- transgene is flanked by flanking
- the recombinant viral genome of the invention is embedded in the
- a "recombinant virus” is a virus derived from the recombinant viral
- the recombinant virus may comprise a transgene, may be an attenuated, replication-competent virus without a transgene, may be a
- the recombinant virus comprising a transgene is capable
- a “flanking element” or “flanking nucleic acid” is a nucleic acid sequence generally derived from a mammalian virus which, when located in positions flanking a transgene, permits the packaging of the transgene into a
- Flanking elements may be the naturally-occurring flanking
- virus or may be artificial nucleic acid elements, e.g. mutated sequences of flanking
- inverted terminal repeats (ITRs) of AAV or Ad include, without limitation, the inverted terminal repeats (ITRs) of AAV or Ad, the long terminal repeats (LTRs) of retrovirus, the " ⁇ " or packaging sequence of herpes
- HSV simplex virus
- 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 may also encode a protein, polypeptide or peptide that is useful for
- transgene may not encode a protein but rather be
- antisense molecule used as an antisense molecule, ribozyme or other regulatory nucleic acid to inhibit
- “Expression control sequences” are nucleic acid sequences that regulate the expression of a gene by being operably linked to the gene of interest.
- “Operably linked” sequences include both expression control sequences that are
- Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer
- RNA processing signals such as splicing and polyadenylation
- carrier vector means a nucleic acid molecule
- mammalian sources derived from mammalian sources, mammalian viral sources, nonmammalian sources,
- nonmammalian viral sources The nonmammalian viral nucleic acid backbone
- nucleic acid sequences inserted into the carrier vector are inserted into the carrier vector.
- a “carrier virus” is an encapsidated carrier vector capable of binding
- ligand nucleic acid means a nucleic acid which
- the nucleic acid encoding the protein may be operably
- Helper function nucleic acid is one or more nucleic acid sequences that encode one or more proteins, peptides or polypeptides, or that is transcribed to
- RNA wherein the one or more proteins, peptides, polypeptides or RNAs are
- helper functions may be naturally-occurring helper functions or may be sequences that have been
- helper viruses may be derived from helper viruses or may be naturally-occurring or artificial
- nucleic acid sequences that encode non-viral proteins that act as helper functions for
- RNA or which encode the proteins, polypeptides or peptides may be operably linked to expression control sequences that regulate the expression of the nucleic
- Replication and/or encapsidation nucleic acid is a nucleic acid
- sequences which encode proteins or polypeptides that are required for replication and encapsidation of the recombinant virus.
- the sequences may be
- nucleic acid sequences encoding the proteins may be any nucleic acid sequences encoding the proteins.
- a "replicon” is an episomal replication origin and those necessary
- the Carrier Vector (or DNA encoding these proteins) to initiate nucleic acid replication.
- the Carrier Vector (or DNA encoding these proteins) to initiate nucleic acid replication.
- the carrier vector of the invention is a chimeric vector backbone
- the carrier vector derived from the nucleic acid of a nonmammalian virus.
- the carrier vector also includes one or
- nucleic acid providing for expression of a protein which can interact with a mammalian cell, replication and/or encapsidation nucleic acid required to replicate
- helper virus functions nucleic acids.
- the carrier vector comprises an
- the recombinant viral genome may comprise a transgene with associated expression regulatory sequences, wherein the transgene and regulatory
- sequences are bordered by flanking elements of a mammalian virus Alternatively,
- the recombinant viral genome does not contain a transgene but rather contains
- the carrier vector comprises the
- carrier vector additionally comprises a ligand nucleic acid providing for expression of a protein which can interact with a mammalian cell
- ligand nucleic acid providing for expression of a protein which can interact with a mammalian cell
- the ligand nucleic acid encodes a protein which can bind to a specific mammalian cell receptor
- the carrier vector comprises the
- carrier virus comprising the carrier vector is to be used to infect a cell line which expresses replication and encapsidation proteins for a recombinant AAV virus (e g ,
- the carrier vector would comprise the
- the carrier vector would comprise the
- the retrovirus is a lentivirus, one or more of the nucleic acids encoding regulatory or
- auxilliary proteins e g , tat, rev, ne vpr, vpu
- the carrier virus is to be used to
- the earner vector would comprise the embedded
- nucleic acid sequences required for its replication and encapsidation The type of nucleic acid sequences required for
- any carrier vector genome may be any carrier vector genome.
- ligand nucleic acid to increase infection by the carrier virus of
- inserts may be carried on separate carrier vectors, but in the most preferred embodiment, the embedded recombinant viral genome and all other desired nucleic acids
- the inability of the carrier vector to replicate in mammalian cells is overcome by supplying a mammalian
- cells infected by the carrier vector maintain a sufficient copy number of the carrier vector extrachromosomally throughout a population of proliferating and dividing
- the chimeric carrier vector is constructed from a backbone of a
- the backbone need not be the entire genome of the nonmammalian virus, but may be only that portion of the genome necessary for
- the vector backbone is derived
- viruses that may be used to form the backbone of the chimeric vector
- the invertebrate DNA virus is a baculovirus In a more preferred embodiment, the invertebrate DNA virus is a baculovirus In a more preferred embodiment, the invertebrate DNA virus is a baculovirus In a more preferred embodiment, the invertebrate DNA virus is a baculovirus In a more preferred embodiment, the invertebrate DNA virus is a baculovirus In a more
- the bacuolovirus is a Granulovirus or Nucleopolyhedrovirus
- the nonmammalian viral backbone is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-ammalian viral backbone
- the nonmammalian virus backbone must be reconstituted from a mammalian virus backbone.
- the baculovirus virus backbone exemplified herein replicates only in insect cells
- transgene but rather has a mutation or deletion in a viral gene and is to be used as a
- vaccine e.g., an attenuated and replication-proficient recombinant virus or a
- the recombinant virus may be any virus of
- Preferred recombinant viruses for delivery of a transgene include adenoviruses,
- retroviruses retroviruses, adeno-associated viruses, herpesvirus amplicons and hepatitis B
- the method of the present invention begins with a desired transgene, then associates
- transgene with appropriate expression regulatory sequences (ERS), e.g., ERS
- an optional spacer or "stuffer” sequence may be inserted in order to
- composition of the transgene sequence depends upon the
- transgene sequence comprises a reporter or marker sequence, which upon
- GFP fluorescent protein
- CAT bacterial chloramphenicol acetyltransferase
- firefly luciferase eukaryotic membrane bound proteins including, for example, CD2,
- CD4 CD4, CD8, the influenza hemagglutinin protein, and others well known in the art, to
- fusion proteins comprising a membrane bound protein appropriately fused to an
- antigen tag domain from, among others, hemagglutinin or myc.
- the presence of a recombinant virus is detected by assays for ⁇ -galactosidase activity.
- the transgene is luciferase
- the recombinant virus gene is luciferase
- the transgene is a non-marker gene which can
- the transgene may be selected from a wide variety of gene products useful
- RNAs in biology and medicine, such as proteins, antisense nucleic acids (e.g., RNAs), or
- catalytic RNAs The invention may be used to correct or ameliorate gene
- transgene sequence is a therapeutic gene which expresses a desired corrective gene product in a host cell.
- therapeutic nucleic acid sequences typically encode products which, upon selection
- transgene may encode any product desirable for study.
- the selection of the transgene sequence is not a limitation of this invention. Choice of a transgene
- the invention also includes methods of producing recombinant virus
- 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 recombinant virus expressing each of the different subunits
- transgene may be encoded by the same transgene In this case, a single transgene would
- IRES internal ribosome entry site
- IRES elements from two members of the picornavirus family have
- IRES elements can be linked to heterologous molecules
- each open reading frame is accessible to ribosomes for efficient translation
- multiple genes can be
- the insert size can be no greater than approximately 4 8
- insert size is approximately 28 kilobases.
- Useful gene products include hormones and growth and
- differentiation factors including, without limitation, insulin, glucagon, growth
- GH parathyroid hormone
- PTH parathyroid hormone
- GRF growth hormone releasing factor
- 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 bone morphogenic proteins
- nerve growth factor nerve growth factor
- BDNF brain-derived neurotrophic factor
- neurotrophins NT-3, NT-4/5 and NT-6 neurotrophins NT-6, ciliary neurotrophic factor (CNTF), glial growth factor (NGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3, NT-4/5 and NT-6, ciliary neurotrophic factor (CNTF), glial
- GDNF derived neurotrophic factor
- neurtuin neurtuin
- persephin agrin
- HGF hedgehog factor
- ephrins noggin
- sonic hedgehog tyrosine hydroxylase
- cytokines and lymphokines such as thrombopoietin (TPO), interleukins (IL) IL-l ⁇ , IL-l ⁇ , TL-2, LL-3, IL-4, IL-5, IL-6,
- IL-J IL-8 IL-9, IL-10, ⁇ L-11, IL-12, LL-13, IL-14, IL-15, IL-16, and IL-17
- monocyte chemoattractant protein MCP-1
- leukemia inhibitory factor LIF
- GM-CSF granulocyte-macrophage colony stimulating factor
- G-CSF G-CSF
- M-CSF monocyte colony stimulating factor
- Fas Fas
- TNF ⁇ and TNF ⁇ tumor necrosis factors and ⁇
- IFN interferons
- IFN- ⁇ and IFN- ⁇ stem cell factor, fTk-2/flt3 ligand.
- Gene products produced by the immune system are also encompassed by this invention. These include, without
- 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
- MCP membrane cofactor protein
- DAF decay accelerating factor
- CR1 CR2
- Still other useful gene products include any one of the receptors for
- receptors examples include flt-1, flk-1, TIE-2,
- TrkA TrkA
- MuSK MuSK
- Eph PDGF receptor
- EGF EGF
- TGF ⁇ receptors TGF ⁇ receptors, the interleukin receptors, the interferon receptors, serotonin
- GDNF receptors ⁇ -adrenergic receptors, ⁇ -adrenergic receptors, the GDNF receptor, p75
- 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 juif f s, max, mad, serum response factor (SRF), AP-1, AP-2, myb, MRG1,
- 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,
- glucose-6-phosphatase porphobilinogen deaminase
- factor VII factor VIII
- factor IX factor II
- factor V factor X
- factor XII factor XI
- von Willebrand factor factor VII, factor VIII, factor IX, factor II, factor V
- factor X factor XII
- von Willebrand factor von Willebrand factor
- urokinase urokinase
- urokinase urokinase
- plasminogen activator plasminogen activator
- heparin cofactor II activated protein
- insulin beta-glucosidase
- pyruvate carboxylase hepatic phosphorylase
- 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, and suicide genes
- thymidine kinase such as thymidine kinase and cytosine deaminase.
- polypeptides such as chimeric or hybrid polypeptides or polypeptides having a non- naturally occurring amino acid sequence containing insertions, deletions or amino acid substitutions.
- polypeptides such as chimeric or hybrid polypeptides or polypeptides having a non- naturally occurring amino acid sequence containing insertions, deletions or amino acid substitutions.
- single-chain engineered immunoglobulins could be useful in certain immunocompromised patients
- Other useful proteins include
- truncated receptors can be used to antagonize the function of their respective
- non-naturally occurring gene sequences include antisense molecules and
- catalytic nucleic acids such as ribozymes, which could be used to reduce
- transgenes include those that encode antigemc peptides
- transgenes can be used for genetic immunization
- Useful transgenes include those
- SIV human T-cell leukemia viruses I and II
- HTLV-I and HTLV-II human T-cell leukemia viruses I and II
- hepatitis A, B, C, D and E pseudorabies virus, rabies virus, cytomegalovirus
- respiratory syncytial virus parainfluenza virus types 1-4, mumps virus, rubella virus,
- polio virus rubeola virus
- influenza virus types A, B and C polio virus, rubeola virus, influenza virus types A, B and C, rotavirus, herpes
- hantavirus adenoviruses, chlamydia pneumoniae, chlamydia trachomatis,
- mycoplasma pneumoniae mycobacterium tuberculosis, atypical mycobacteria, feline
- leukemia virus feline immunodeficiency virus, bovine immunodeficiency virus,
- transgenes may also be directed against peptides from tumor antigens to provide immunization for tumors
- helper functions and the ligand The choice of expression control sequence depends on
- sequences typically include a promoter, an enhancer, such as one derived from an immunoglobulin gene, SV40, cytomegalovirus, etc., and a polyadenylation sequence which may include splice donor and acceptor sites.
- the polyadenylation sequence typically includes a promoter, an enhancer, such as one derived from an immunoglobulin gene, SV40, cytomegalovirus, etc., and a polyadenylation sequence which may include splice donor and acceptor sites.
- 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
- SV-40 T intron sequence also derived from SV-40, and is referred to as the SV-40 T intron sequence.
- IVS internal ribosome entry site
- An IRES sequence is used to produce more than one polypeptide from a single gene transcript.
- An IRES sequence can be used for the
- high-level constitutive expression will be desired
- promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter/enhancer, the cytomegalovirus (CMV)
- PGK phosphoglycerol kinase
- inducible promoters may be desired
- Inducible promoters are those which are regulated by exogenously supplied
- MMTV mammary tumor virus
- T7 polymerase promoter system [WO 98/10088]
- ecdysone insect promoter [No et al, Proc Natl Acad Sci
- promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, or in replicating cells
- the transgene is under the control of the native p5 promoter of AAV
- nucleic acid sequence of interest will be used 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
- genes include the promoters from genes encoding skeletal ⁇ -actin, myosin light chain 2A,
- liver [albumin, Miyatake et al J Virol , 71 5124-32 (1997), hepatitis B virus core
- host cell of choice may be selected by one of skill in the art using the guidance
- nonmammalian vector of the instant invention After following one of the methods for producing and packaging the recombinant vector as taught in this specification
- transgene in the cell may be monitored by Southern blotting or quantitative PCR, the level of RNA expression may be monitored by Northern blotting or quantitative
- RT-PCR and the level of protein expression may be monitored by Western blotting
- nucleic acid molecule into a vector of the instant invention.
- nucleic acid sequence encoding a ligand produces a carrier virus which is able to
- recombinant virus one may infect mammalian cells with the recombinant virus, then
- Flanking elements are required for replication, excision and
- these elements flank the viral genes when the viral
- DNA integrates into a host cell chromosome In the case of integrating viruses,
- extrachromosomal viruses e g HSV
- flanking sequences e g HSV
- the recombinant virus is a
- recombinant adenovirus comprises a selected transgene operably linked to
- Adenoviral flanking elements are ITRs and are 100-200 bp in length A large number of
- adenoviral flanking elements are known, such as those from human adenoviruses types 1 -46, chimpanzee adenoviruses, canine adenoviruses, bovine adenoviruses [all
- the recombinant virus is a recombinant
- retrovirus and comprises a selected transgene operably linked to expression
- flanking elements are:
- LTR long terminal repeat
- LTRs contain strong promoter and enhancer sequences
- the recombinant virus is
- AAV a recombinant AAV, and comprises a selected transgene operably linked to expression regulatory sequences and AAV flanking elements
- AAV ITRs consist of approximately 145 bp at the 5' and 3' ends of the
- AAV genome The AAV ITRs are required for replication, excision and encapsidation of both wild type and recombinant AAV virions
- the recombinant virus is either a herpesvirus
- flanking elements would be the viral
- HSV amplicons are defective HSV genomes containing the packaging sequence ( ), viral origin of DNA
- helper herpesvirus or substitute helper functions the amplicon is replicated and packaged as head-to-tail
- the recombinant virus is a recombinant
- HBV hepatitis B virus
- nonmammalian viruses are not infectious to mammalian cells
- nonmammalian virus the nonmammalian virus may be modified by incorporating
- the nonmammalian backbone may
- ligand DNA also be modified by incorporation of ligand DNA to increase infection of mammalian host cell by the nonmammalian virus.
- DNA by the subsequently produced nonmammalian virus will permit infection or mcrease infection of mammalian cells
- present invention is modified by addition of DNA encoding components needed to
- the ligand DNA is selected from genes which, when expressed, yield
- the nonmammalian expression regulatory sequence may be identical, similar,
- the expression regulatory sequences comprise a promoter derived from the native
- nonmammalian virus from which the nonmammalian vector backbone is derived In
- the promoter is polyhed ⁇ n early promoter (polH)
- nonmammalian vector backbone is derived from baculovirus
- the ligand DNA will not be expressed when the
- chimeric vector infects the mammalian host cell The absence of expression of the
- ligand encoded by the DNA may be useful to prevent incorporation of ligand mto
- the recombinant virus coat encoded from the carrier nonmammalian vector, which could disrupt its structural integrity or cause adverse immunogemc reactions in an animal
- ligand expression is desired in the mammalian host cell
- ligand DNA is expressed because the nonmammalian expression regulatory
- sequences are also activated in the mammalian cells
- the ligand DNA can be essentially any nucleic acid that encodes a
- the ligand can be naturally- occurring protein, a fragment of a naturally-occurring protein that has a desired
- the ligand can be one of general specificity, which
- VSV-G virus glycoprotem G gene
- BSV bovine syncytial virus envelope
- glycoprotem gene or amphotropic envelope gene as illustrated below, or it may be
- ligand may cause the virus to bind via electrostatic interactions or other general
- Useful ligand nucleic acids may be any nucleic acid which encodes a ligand that permits the nonmammalian virus to interact with the mammalian cell
- the ligand may be one which increases the electrostatic interaction between the virus and the mammalian cell for a receptor found on the mammalian
- nucleic acids include, without limitation, nucleic acids encoding peptide hormones, growth
- nucleic acids useful as a ligand include all those
- nucleic acid may encode PDGF, EGF, bFGF, aFGF, insulin, IGF-I, IGF-II, apoE,
- the ligand nucleic acid may encode a
- immunoglobulin e g , ScFv, chimeric immunoglobulin, humanized immunoglobulin, etc
- MHC molecule that
- ligand nucleic acids of interest encode a member of the extracellular matrix such as
- ligand which is normally secreted may be modified by incorporating a nucleic acid
- the anchoring domain is a region that secures the ligand in
- the anchoring domain is at the 3' end of
- the coding sequence for the ligand in a further preferred embodiment, the
- anchoring domain is derived from a viral coat protein, such as HIV gp41 (which
- anchors gpl20 coat protein to the viral envelope Other examples include E protein of dengue virus or the 14 kDa protein of vaccinia virus
- the ligand nucleic acid also may encode a protein that is normally
- type of ligand nucleic acid include a number of the CD antigens, such as the T cell
- TCR tumor necrosis factor receptor
- CTLA-4 receptor CTLA-4 receptor
- B-7 integrins such as Mac-1, LFA-1, and
- intercellular adhesion molecules such as ICAM-1, ICAM-2, ICAM-3 and
- the ligand may also be an artificial or mutated counter-receptor, such as a cell-surface
- the ligand is one that is normally present on a
- gpl20 of HIV virus and which mediates binding to a mammalian cell, for example, gpl20 of HIV
- the ligand is one that is normally
- Protein A from Staphylococcus aureus is known to bind to
- the mammalian host cell is genetically
- binding of the carrier virus to the mammalian host cell For example, one may
- a mammalian host cell line to express a growth factor receptor, such as the
- EPO receptor design the carrier vector to comprise a ligand nucleic acid
- the ligand DNA is the VSV-G gene This gene may be placed under the control of the baculovirus polyhedrin (pPH) early
- VSV-G protein when expressed, modifies the mature carrier virus
- the ligand DNA is the
- BSV env gene which functions in the context of the invention in a similar manner.
- the present invention exploits the
- the ligand DNA is a gene which expresses an asialoglycoprotein, which
- mammalian lectins e.g., the hepatic asialoglycoprotein receptor
- recombinant virus is a retrovirus, then the replication and encapsidation functions
- the carrier vector or the mammalian host cell may comprise nucleic acids encoding those replication and encapsidation
- Mammalian host cells such as
- PCT US98/19463 express AAV rep and cap genes for replication and packaging
- retroviruses have been constructed [see, e.g., Cone et al., Proc. Natl. Acad. Sci.
- herpesviruses see, e.g., U.S. Pat. No. 5,851,826, are also known.
- a cell line comprises all the necessary replication and encapsidation
- the carrier vector need not comprise any replication and/or encapsidation
- the cell line may comprise the necessary replication and
- cell line may express the replication and encapsidation functions constitutively or inducibly Constitutive or inducible expression may be controlled by using any of
- replication and encapsidation functions is inducible.
- the replication and encapsidation functions is inducible.
- the replication and encapsidation functions are stably transfected or
- a mammalian cell line used in the instant invention may comprise none of the functions required for replication or encapsidation, or may comprise
- mammalian cell line comprises none of the functions required for replication or
- these functions must be introduced into the cell by a vector for production of the recombinant virus.
- a vector for production of the recombinant virus In a preferred embodiment, one or more
- carrier viruses of the instant invention are used to transduce the mammalian cell line
- a single carrier virus comprising the replication and
- a single carrier virus comprising the replication and
- nucleic acid sequences required for recombinant virus production are used to transduce the mammalian cell line.
- the mammalian cell line comprises some of the replication or encapsidation functions, these functions must be introduced into the cell by a vector
- one or more of the following viruses are provided for production of the recombinant virus.
- one or more of the following viruses are provided.
- carrier viruses are used to transduce the mammalian cell line with the nucleic acids
- a single carrier virus comprising the missing replication
- encapsidation functions are used to transduce the mammalian cell line.
- a single carrier virus comprising the missing replication
- nucleic acid sequences required for recombinant virus production are used to generate nucleic acid sequences required for recombinant virus production.
- recombinant virus differ depending upon the type of recombinant virus. In general, the required replication and encapsidation functions are known in the art for the
- recombinant viruses various recombinant viruses.
- recombinant vectors In preferred embodiment of recombinant vectors,
- gag,pol and env and tat, rev and nef Tor
- lentiviruses lentiviruses
- recombinant adenoviruses require all of part of the functions encoded
- herpesviruses require a large number of genes, which may be provided
- helper herpesvirus or by a carrier vector comprising the required herpesvirus
- the replication and encapsidation functions are encoded by nucleic acids encoding the naturally-occurring proteins having the
- replication and encapsidation functions In another embodiment, the replication and
- encapsidation functions are encoded by nucleic acids encoding fragments or mutems
- recombinant viruses may be produced using nucleic acids encoding the appropriate
- the rep and cap sequences are regulated by a native AAV p5 promoter
- nucleic acid sequences encoding the replication and encapsidation are desired, the nucleic acid sequences encoding the replication and encapsidation
- adenovirus functions for adenovirus are regulated by their native adenovirus promoters Native promoters may also be used for regulating the expression of replication and
- carrier vector is that the replication and encapsidation functions are only expressed
- carrier virus has an embedded recombinant viral genome comprising a transgene
- the expression of the rep and cap genes is regulated by their
- the carrier viruses contain nucleic acid sequences that encode replication and encapsidation functions.
- the carrier viruses contain nucleic acid sequences that encode replication and encapsidation functions.
- nucleic acid sequences encoding the replication and encapsidation are provided.
- helper functions may also be
- helper function may differ depending upon the type of recombinant virus and/or the amount of genome that has been deleted. Helper functions include viral proteins,
- AAV requires helper functions from adenovirus or herpesvirus or
- helper functions are required for high levels of recombinant virus production.
- recombinant virus One may then transduce the mammalian host cells with various
- nucleic acids encoding potential helper functions may be any nucleic acid that is known or thought to encode a helper function.
- helper functions may be any nucleic acid that is known or thought to encode a helper function.
- the helper function is one or more viral proteins. In a more preferred
- helper virus proteins are insufficient to produce a mature helper
- helper virus comprises a recombinant AAV genome
- the helper functions are nucleic acids derived from a virus
- helper functions are derived from adenovirus
- the helper functions are at least El a, Elb and E2a from adenovirus, and may also include E4ORF6 and VAI
- the nucleic acid encodes the
- helper functions from the helicase-primase complex of HSV (UL5, UL8 and UL52)
- HSV DNA replication genes (UL5, 8, 52, 29, 30, 9
- helper functions for recombinant AAV may be provided by chemical or physical agents, including ultraviolet light, cycloheximide, hydroxyurea
- 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
- the transfection or infection may be stable or
- the mammalian cell line may stably express (either on an
- helper functions may be expressed by the helper functions
- mammalian cell line while other helper functions are introduced by a vector.
- 293 cells constitutively produce adenoviral El a and
- the helper functions required for the production of infectious recombinant AAV are introduced into the host cell by transfection or infection of a vector.
- the helper functions are transduced into the mammalian cells by a carrier virus. In a more preferred embodiment, some or all of the helper functions are transduced into the mammalian cell by a carrier virus
- all of the helper functions are transduced into the mammalian cell by a carrier virus comprising the embedded recombinant viral genome, any required
- the carrier vector has a baculovirus backbone.
- ribosome entry site (IRES) sequence may be placed between E2A and E4orf6 if
- helper function gene may be supplied with its own promoter. These genes may be
- helper functions are provided on the carrier vector itself
- the promoters regulating those genes may be constitutive or inducible.
- helper functions may be regulated by any of
- the expression regulatory sequences may provide
- the native promoter of the helper function is the native promoter of the helper function
- an inducible promoter of a helper of a helper
- a constitutive promoter of a helper function protein is used.
- the a constitutive promoter of a helper function protein is used.
- constitutive promoter is the CMV promoter. In another preferred embodiment, one
- constitutive promoters are used for certain helper function proteins, and one or more native promoters are used for other helper function proteins.
- each protein or polypeptide required for helper is independently selected from the amino acid or polypeptide required for helper
- nucleic acid whose expression is regulated by its own
- nucleic acid is transcribed to a single
- an IRES may be placed between the coding sequences of
- polycistronic mRNA If only a single polycistronic transcript is produced, only a single promoter, optional enhancer, and polyadenylation signal are required for
- the helper function may also encode the helper function by using both monocistronic mRNAs that
- the carrier vector in a preferred embodiment of the instant invention, the carrier vector
- helper functions comprise adenovirus El a, Elb
- E4ORF6 and VAI preferably include E4ORF6 and VAI.
- helper functions are encoded by a single polycistronic transcript, and the promoter for the helper functions is a constitutive promoter, preferably the CMV promoter.
- the promoter for the helper functions is a constitutive promoter, preferably the CMV promoter.
- Other recombinant viruses would require different helper functions
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Abstract
This invention relates to nonmammalian carrier vectors and viruses useful in the production of high titers of recombinant viruses which may contain foreign DNA inserts or which may be point-mutated or deleted viruses, and methods of producing those viruses. The nonmammalian carrier vector ('carrier vector') is a chimeric vector which includes those portions of a nonmammalian virus backbone which allow replication in a nonmammalian host cell. The carrier vector includes various nucleic acid cassettes, which may include an embedded recombinant viral genome containing a desired transgene, components necessary for production of a replication-defective recombinant virus containing the transgene, and domains that permit the carrier vector to bind to mammalian cells. The invention also provides methods of producing high concentrations of recombinant virus as a substantially homogeneous preparation, compositions to produce the recombinant virus, and recombinant viruses.
Description
WO 00/73480 PCT/USOO/l4481
COMPOSITIONS AND METHODS FOR PRODUCTION OF RECOMBINANT VIRUS USING A CARRIER VECTOR DERIVED FROM A NONMAMMALIAN VIRUS
TECHNICAL FIELD OF THE INVENTION
This invention relates to novel nonmammalian carrier vectors and 5 viruses useful in the production of high titers of recombinant viruses which may
contain foreign DNA inserts or which may be point-mutated or deleted viruses, and
methods of producing those viruses. The nonmammalian carrier vector ("carrier
vector") is a chimeric vector which includes those portions of a nonmammalian
virus backbone which allow replication in a nonmammalian host cell. The carrier
0 vector includes various nucleic acid cassettes, which may include an embedded
recombinant viral genome containing a desired transgene, components necessary for
production of a replication-defective recombinant virus containing the transgene,
and domains that permit the carrier vector to bind to mammalian cells. The invention also provides methods of producing high concentrations of recombinant
5 virus as a substantially homogeneous preparation, compositions to produce the
recombinant virus, and novel recombinant viruses.
BACKGROUND OF THE INVENTION
A recombinant virus carrying a foreign DNA insert may be used to
deliver genes to cells, where the gene may be expressed, if desired, to permit
production of recombinant proteins in vitro or in vivo, vaccination of human and
non-human mammals, or treatment or amelioration of diseases or genetic defects in
humans or non-human mammals. One may treat or ameliorate diseases or genetic
defects by providing normal gene products, increased levels of gene products or by blocking endogenous production of a gene, whose expression would be deleterious
to the cell or organism.
Methods for delivering an exogenous gene to a mammalian cell
include the use of mammalian viral vectors, such as those which are derived from
retroviruses, adenoviruses, herpes viruses, vaccinia viruses, polio viruses, adeno-
associated viruses, hybrid viruses (e.g., hybrid adenovirus-AAV, see U.S. Pat. No.
5,856, 152) and the like. Other methods include direct injection of DNA, biolistic
administration of DNA, electroporation, calcium phosphate precipitation, as well as
methods of administration which utilize ligand-DNA conjugates, liposome
conjugates of DNA, polycation-DNA complexes or adenovirus-ligand-DNA
conjugates.
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. Alternatively, the transgene may be one that is
useful for corrective applications. For instance, a transgene may be a normal gene
that replaces or augments the function of a patient's defective gene The transgene
may be one that counteracts the effects of a disease, such as introduction and
expression of a gene that is distinct from the one that it replaces or augments, but
which has the same function or compensates for the defective gene's function. The
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 corrective effect A transgene may also be used for immunization against various
agents, by provoking an immunogenic response in an animal. Delivery of
therapeutic transgenes to a patient thus effects a correction of a defect or
prevention of disease. The transgene also may be one which is useful for production of proteins in vitro, such as for large-scale production of therapeutic
proteins
Appropriate genes for expression in the cell include, without
limitation, those genes which are normally expressed in cells but whose products are
produced in insufficient amounts. Alternatively, the appropriate gene for expression
is one which expresses a normal gene product which replaces a defective gene
product, encodes ribozymes or antisense molecules which repair or destroy mutant
cellular RNAs expressed from mutated genes, or modifies or destroys viral RNAs
Transgenes used for production of proteins in vitro include proteins such as secreted factors, including hormones, growth factors and enzymes.
Many gene therapy methods involve supplying an exogenous gene to
overcome a deficiency in the expression of a gene in a patient. Some of these
deficiencies are congenital and are due to a mutation in a particular gene in all the
cells of the patient For instance, in cystic fibrosis, there are one or more mutations
in the gene encoding the cystic fibrosis transmembrane conductance regulator
(CFTR) which prevents the CFTR protein from functioning properly In other
cases, a deficiency in gene expression is due to an accident or disease that occurs
during the patient's life For instance, in Type I diabetes mellitus, the β pancreatic
islet cells, which produce insulin, are destroyed, such that patients with this disease
can no longer synthesize insulin In other cases, the endogenous gene may be
structurally normal but is not produced in high enough quantities due to disease, medical treatment or other environmental conditions, or mutations in the regulatory
elements of the endogenous gene. For example, there are a number of blood
disorders, such as anemia, in which there is insufficient production of red blood
cells, which may be treated with erythropoietin (EPO) or with a transgene encoding
EPO Transgenes may also be used for genetic immunization, i.e., to elicit an
immune response to a pathogen in an animal, including humans. For instance, a
transgene may include a sequence from a viral, bacterial or fungal pathogen, such as
influenza virus, human immunodeficiency virus (HIV), or mycobacterium
tuberculosis.
Certain methods are amenable to targeted delivery of the exogenous gene to specific tissues, such as liver tissue. One method of delivering genes to specific cells relies upon the function of a cell-specific receptor. The
asialoglycoprotein receptor (ASGP-R), which is present on the surface of
hepatocytes (Spiess et al., 1990, Biochem. 29: 10009-10018), is a lectin which has
affinity for the terminal galactose residues of glycoproteins, and has been used to
target gene delivery to liver hepatocytes. For example, a DNA complex is bound to
a ASGP-R on the cell surface, allowing subsequent endoyctosis by the liver
hepatocyte
Viruses that are commonly used in gene delivery applications are
modified by replacing viral nucleic acid with a desired transgene Frequently, DNA
removed from the virus encodes proteins necessary for viral replication or
encapsidation, in which case the recombinant virus containing a transgene is
replication-deficient and will not replicate or encapsidate in the host To permit
replication and encapsidation, current methods recognize that those portions of
D A which have been deleted must be supplied by wild-type or modified viruses or
by plasmids containing DNA encoding the required gene products Supplying wild-
type or modified virus may result in recombinant virus stocks contaminated with wild-type or modified virus Supplying plasmids encoding the required gene
products through cotransfection results in low efficiency of recombinant virus
production, as well as recombination events which yield wild-type virus
contaminants.
A number of different viruses have been used to deliver a transgene
to mammalian cells These viruses include retrovirus, hepatitis B virus (HBV),
adenovirus, adeno-associated virus (AAV) and herpesvirus. AAV possesses unique
features that make it attractive as a vector for delivering foreign DNA (i.e , a transgene) to cells, and various groups have studied the potential use of AAV in the treatment of disease states.
AAV is a parvovirus, the genome of which is about 4.7 kb in length,
including 145 nucleotide inverted terminal repeats (ITRs). The AAV genome
encodes two genes, rep and cap, each of which expresses a family of related
proteins from separate open reading frames and produced as a result of alternative
mRNA splicing. Rep polypeptides (repl , rep68, rep52, and rep40) are involved in
replication, rescue and integration of the AAV genome. Cap proteins (VP1, VP2,
and VP3) form the virion capsid. Flanking the rep and cap open reading frames at
the 5' and 3' ends of the AAV genome are the 145 bp ITRs, the first 125 bp of
which are capable of forming Y- or T- shaped duplex structures. The entire nucleic
acid encoding rep and cap can be excised and replaced with a transgene [B. J.
Carter, in "Handbook of Parvoviruses", ed. , P. Tijsser, CRC Press, pp.155-168
( 1990)] . The ITRs represent the minimal sequence required for replication, rescue,
packaging, and integration of the AAV genome.
When this nonpathogenic human virus infects a human cell, the viral
genome integrates into chromosome 19 resulting in latent infection of the cell.
Production of infectious virus and replication of the virus does not occur unless the
cell is coinfected with a lytic helper virus, such as adenovirus (Ad) or herpesvirus.
Upon infection with a helper virus, the AAV provirus is rescued and amplified, and
both AAV and helper virus are produced. The infecting parental ssDNA is
converted to duplex replicating form (RF) DNAs in a rep dependent manner. The
rescued AAV genomes are packaged into preformed protein capsids (icosahedral symmetry approximately 20 nm in diameter) and released as infectious virions that
have packaged either + or - ss DNA genomes following cell lysis. However,
progress towards establishing AAV as a transducing vector for the delivery of DNA
in the form of a desired transgene has been slow for a variety of reasons.
Replacing the rep and cap sequences with a desired transgene yields
a recombinant virus capable of delivering the transgene to target host cells.
However, because AAV requires a particular genome packaging size, addition of a
transgene results in deletion of necessary gene functions for rep and cap. In current
methods, necessary gene functions replaced by the transgene are supplied by viruses
or additional plasmids. Furthermore, the requirement by AAV for helper virus
functions also requires the use of helper viruses (either wildtype or crippled viruses)
or plasmids containing the helper virus functions.
One method that has been used to produce recombinant AAV
(rAAV) vectors comprises co-transfecting eukaryotic cells with a plasmid
containing rAAV (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). See U.S. Pat. No. 5,753,500. The disadvantage of this method is that the rAAV vector stock is contaminated with helper virus, which is labor-intensive and
difficult to separate from the helper virus, and co-transfection of two plasmids along
with infection by a helper virus is inefficient and cannot be easily scaled up for industrial production of rAAV.
A second method that has been used to produced rAAV involves a triple plasmid transfection of eukaryotic cells. In this method, one 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 functions,
i e adenoviral genes such as El a, El b, E2a and E4 (the helper plasmid) The
disadvantage of this method is that a triple transfection is also inefficient and is
difficult to scale up
A third method involves the use of a packaging cell line such as one
including AAV functions rep and cap. See U S Pat No 5,658,785 and U S Serial
No PCT US98/19463 The packaging cell line may be transfected with a cis
plasmid comprising the transgene and ITRs, and infected by wild-type adenovirus (Ad) helper See U S Pat No 5,658,785 Alternatively, the packaging cell line
may be co-infected by a hybrid Ad/ AAV, in which a hybrid Ad vector caries the cis
plasmid in the El locus (see U S Pat. No. 5,856,152), and by a wild-type or mutant
Ad that supplies El The disadvantage of this method is that wild-type Ad may be
produced, which must be separated from the rAAV vector before use in a patient
Thus, current methods of producing recombinant AAV are incapable of yielding the high amounts of essentially homogeneous virus for pharmaceutical
compositions needed for the treatment of a large number of patients in a easily
scaled industrial production.
Nonmammalian viruses have been used to transiently express particular individual exogenous proteins in either mammalian or non-mammalian
cells For example, viruses of the family Baculoviridae, or "baculoviruses", which
normally infect members of the order Lepidoptera, have been used to express
exogenous genes in insect cells Baculoviruses have also been reported to enter mammalian cells, and baculoviral DNA has been detected in nuclear extracts of
mammalian cells (Volkman et al, 1983, Appl. Environ. Microbiol. 45' 1085-1093)
While one report of baculovirus-mediated gene expression in mammalian cells has
appeared, the authors later attributed the apparent reporter gene activity to the
reporter gene product being carried into the cell after a prolonged incubation of the
cell with the virus (Carbonell et al , 1987, Appl Environ Microbiol 53 1412-
1417) These authors reported that, when the exogenous gene gains access to the
cell as part of the baculovirus genome, the exogenous gene is not expressed de
nova Subsequent studies have demonstrated baculovirus-mediated gene expression
of particular proteins in mammalian cells (Boyce et al , 1996, Proc Natl Acad Sci
USA, 93 2348-2352)
While baculovirus has been used for expressing particular proteins in
a mammalian cell, see U S Pat No 5,731,182, baculovirus has not been used to
produce pharmaceutical compositions of replication-deficient recombinant virus
using an easily scaled industrial process As disclosed in U S Pat No 5,731,182,
the genome of the baculovirus may be modified by insertion of ligand DNA, which
comprises a gene encoding a mammalian receptor specific protein that allows the
baculovirus to bind and enter mammalian cells The nonmammalian virus infecting
the mammalian cells allows only for transient expression of the transgene within the
mammalian cell In addition, the methods disclosed in U S Pat No 5,731,182 do
not result in production of an altogether distinct, essentially homogeneous recombinant virus, at high titers
The problem of generating recombinant replication-deficient virus
that is produced in the absence of helper viruses and by an efficient method that is
applicable to large-scale industrial production has not been solved until the present
invention. Current viral production methods include costly and time consuming purification and concentration steps, and are incapable of producing sufficient
recombinant virus for pharmaceutic applications. In the case of AAV, for example,
current methods produce at most on the order of 104-105 genomic copies (gc) of
recombinant virus per producer cell. Similarly, current methods are suitable for
producing recombinant adenovirus in amounts on the order of IO4 particles per
producer cell, and retroviruses in amounts on the order of 102-104 colony forming
units (cfu) per producer cell. Current production methods result in contaminating
helper virus which must be inactivated and/or removed from the final products prior to pharmaceutical application. Thus, there exists an unfulfilled need for a method of manufacturing recombinant mammalian virus at high titers free of other
contaminating virus in order to produce recombinant viruses capable of delivering a
desired transgene to mammalian cells, or immunizing cells against viral or bacterial
infection by the use of such recombinant viruses, in a stable fashion.
SUMMARY OF THE INVENTION
The invention exploits the properties of nonmammalian and
mammalian viruses to create novel chimeric vectors and viruses for the manufacture of an essentially homogeneous recombinant virus preparation in the absence of
contaminating helper virus using a process that may be easily scaled for industrial production. The essentially homogeneous recombinant virus may be used for
various purposes, including delivering a desired transgene to mammalian cells for
pharmaceutic applications including immunization and correction of genetic defects;
transient and stable gene transfer in vivo, in vitro and ex vivo; production of
proteins in vivo or in vitro, and other methods in which high levels of gene
transduction into a cell are required, e.g , in the production of expression libraries
for screening compounds or for introducing genes into cells that are not easily
transfected
The carrier vector of the invention is a chimeric vector backbone
derived from the nucleic acid of a nonmammalian virus, and includes one or more of
the following elements 1) an embedded recombinant viral genome, 2) nucleic acid sequences which encode proteins required for replication and encapsidation of the recombinant virus genome, 3) nucleic acid sequences encoding helper functions (if
the recombinant virus to be produced is helper-dependent, e.g., AAV), 4) nucleic
acid sequences encoding a ligand that can interact with a mammalian cell, and 5)
regulatory control sequences that regulate nucleic acid sequences in the
nonmammalian virus backbone or in a replication-deficient portion or modification
thereof. The carrier vector may also include any other nucleic acid sequences that
are required to produce a replication-deficient recombinant virus.
In one embodiment of the invention, one or more carrier vectors may
comprise all of the elements required to produce a replication-deficient recombinant vector in a particular host cell or cell line. The number and type of elements that
are required will depend upon the particular host cell used and the type of
recombinant vector produced. For instance, if a recombinant AAV vector is desired and the host cell line is one which has rep and cap stably integrated in its genome,
the carrier vector or vectors would comprise 1) an embedded recombinant viral
genome comprising the AAV ITRs and the transgene and 2) separate helper functions, which may include any nucleic acid sequence required for replication and
encapsidation of the rAAV For instance, these helper functions may include any
one or a combination of El, E2a, E4ORF6 and VAI from adenovirus (Ad) If a
recombinant AAV vector is to be produced in a host cell line that does not express
/ ep and cap, then the carrier vector or vectors may also include the DNA sequences
encoding / ep and cap
Alternatively, if a recombinant retrovirus is desired, the carrier
vector or vectors would comprise 1) an embedded recombinant viral genome
comprising the retroviral LTRs and the transgene of interest driven from the
retroviral LTRs or from a heterologous promoter, and 2) DNA sequences encoding anv one or a combination oϊ gag, pol and env for the functions of replication and
encapsidation of the retrovirus not supplied in the host cell In a preferred
embodiment, all of the required elements to produce a recombinant virus in a
particular host cell are contained on a single carrier vector because the use of a
single carrier vector having all functions not supplied by a host cell increases the efficiency of transduction, and can be more easily scaled for industrial production of the embedded recombinant virus
In an alternative embodiment, the earner vector comprises an
embedded recombinant viral genome, and any required replication, encapsidation and/or helper functions are provided by a helper virus or a plasmid
The embedded recombinant viral genome may comprise a transgene and DNA elements required for replication of a mammalian virus The transgene
comprises the gene of interest, regulatory elements to regulate its expression, and
an optional DNA spacer. The transgene is flanked by the DNA elements required
for replication of a mammalian virus, such as the ITRs of AAV, the LTRs of
retrovirus, or the ITRs of adenovirus. The recombinant viral genome is embedded
within the nonmammalian virus backbone, optionally along with one or more of the
other DNA sequences listed above, resulting in a chimeric carrier vector of the
present invention.
In an alternative embodiment, the embedded recombinant viral
genome does not contain a transgene but the recombinant viral genome itself contains point mutations or deletions. In this embodiment, the point mutations or
deletions function to attenuate the replication of the subsequently-produced
recombinant virus. The attenuated recombinant virus may be any virus which could
be useful for vaccination, including, without limitation, picornaviruses such as
poliovirus; hepatitis viruses such as hepatitis B and hepatitis C; cold-adapted
respiratory syncytial virus (RSV); cold-adapted influenza virus; parainfluenza virus types 1, 2 and 3; and rotavirus.
The carrier vector is replication-proficient in its native host cells.
For example, employing a baculovirus backbone results in a chimeric carrier vector
that is replication-proficient in insect cells. In contrast, the embedded recombinant viral genome, optionally containing a transgene, is unable to excise, replicate, and package into virions because its promoters are inactive in insect cells. However,
once the chimeric carrier vector infects a mammalian cell, the essential gene
products required for replication and packaging of the carrier vector in its
permissive native cell are no longer expressed. Thus, the carrier vector does not
replicate in mammalian cells, and instead exists transiently within the mammalian
cell.
In contrast, once the carrier vector has infected a mammalian cell,
the mammalian regulatory sequences within the carrier vector controlling the
embedded recombinant viral genome and other mammalian DNA sequences are
activated, such that the recombinant viral genome is capable of being excised from
the carrier vector and replicated. The capsid proteins which form the capsid of the
recombinant virus are expressed such that the recombinant viral genome is
encapsidated, which yields an infectious recombinant virus. The recombinant virus
is essentially free of carrier vector because the carrier vector is not replicated in
mammalian cells.
In a preferred embodiment, the recombinant virus is replication-
deficient because there are no replication or helper functions present in the newly
formed virions; i.e., the recombinant virus lacks part or all of the coding regions of
the native virus genome. In embodiments of the invention in which the recombinant
virus is helper-dependent, such as rAAV, the recombinant virus lacks both
functional replication and encapsidation functions. In embodiments of the invention
in which the recombinant virus is not helper-dependent, the recombinant virus lacks
functional replication coding regions or other essential genes.
In cases where helper functions are required for recombinant virus production, recombinant virus may be produced without the need for coinfection
and subsequent production of helper virus if a carrier vector includes the necessary
helper functions. Thus, the invention yields lysates of substantially pure and
essentially homogeneous preparations of the particular recombinant virus of interest
in the absence of helper virus.
This invention thus has many advantages over current methods for manufacturing recombinant viruses. These advantages include: (l) the
nonmammalian virus backbone permits insertion of large DNA sequences without
compromising the efficiency of recombinant virus production; (2) sequences
normally toxic to mammalian cells (e.g., AAV rep, VSV-G, retroviral envelope proteins, eukaryotic regulatory proteins, etc.) are not expressed in substantial
amounts from their mammalian regulatory sequences in the nonmammalian host cell
of the nonmammalian carrier vector and thus can be tolerated by the nonmammalian
carrier vector during the course of its replication in the nonmammalian host cell; (3)
nonmammalian viruses do not replicate in mammalian cells, precluding
contamination of the final eukaryotic vector stocks with the nonmammalian carrier
vector; (4) in some embodiments no helper viruses are necessary, with the result
that the final recombinant virus preparation is essentially free of helper virus; (5)
frequency of wildtype virus production due to homologous or non-homologous recombination is minimized; and (6) the methods of the present invention are
particularly suitable to large scale production of recombinant viruses which are
themselves replication-deficient. Additionally, nonmammalian viruses are not
normally pathogenic to mammalian cells, may be propagated in serum free media,
and may be grown to a high titer. Other features and advantages of the invention
will be apparent from the following drawings, the description of the invention and
its preferred embodiments, and the examples described herein.
In one embodiment, the present invention includes nonmammalian
carrier vectors containing elements that are required to produce replication-deficient
recombinant viral vectors. In a preferred embodiment, the nonmammalian carrier
vector contains all the elements required to produce a replication-deficient recombinant viral vector. In an even more preferred embodiment, a single
nonmammalian carrier vector contains all the required elements to produce a
replication-deficient recombinant viral vector. In another preferred embodiment,
the nonmammalian carrier vector is a baculovirus.
In another embodiment, the invention includes a method of
producing replication-deficient recombinant viral vector lysates and stocks that are free of helper or other contaminating virus. In a preferred embodiment, the method
is one which is easily scaled for industrial production of recombinant viral vectors.
In another preferred embodiment, the method is one in which a high titer of
recombinant viral vector lysates and stocks is achieved.
In another embodiment, the invention includes attenuated,
replication-competent recombinant viruses and a method of producing such viruses
free of helper or other contaminating virus. In a preferred embodiment, these
attenuated, replication-competent viruses may be used for immunization.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic diagram of recombinant baculoviruses with
target genes inserted into the loci of either polyhedrin or plO genes
Figures 2A and 2B represent a genetic map of AAV type 2 Figure
2 A is a schematic representation of the viral genome rep encodes replication
proteins (Rep78, Rep68, Rep52, and Rep40) and cap encodes encapsidation
functions (VP1, VP2, and VP3) Right-angled arrows: the p5, pl9, and p40 viral
promoters, downward vertical arrow: common polyadenylation signal upstream of
the 3 '-ITR Figure 2B represents the transcripts derived from each of the three
promoters A,," polyadenylation.
Figure 3 is a schematic diagram of constructed plasmids used in this
invention
Figure 4 shows the steps involved in rAAV production by traditional
adenovirus infection/plasmid co-transfection method (Shenk et al., US patent
#5,436, 146)
Figure 5 shows the steps required for rAAV production through the
use of two recombinant baculoviruses (BV-EiOV-RC and BV-cisEFGFP).
Figure 6 shows the steps required for rAAV production through the
use of stable cell line 293-CG3 together with one recombinant baculovirus (BV- EiOV-RC)
Figure 7 shows the steps required for rAAV production through the
use of stable cell line expressing AAV rep and cap genes together with recombinant
baculovirus (BV-EiOV-cisEFGFP-El).
DET AILED DESCRIPTION OF THE INVENTION
Definitions and General Techniques
Unless otherwise defined, all technical and scientific terms used
herein have the meaning as commonly understood by one of ordinary skill in the art
to which this invention belongs The practice of the present invention employs,
unless otherwise indicated, conventional techniques of chemistry, molecular
biology, microbiology, recombinant DNA, genetics, virology and immunology See, e g , Sambrook et al , 1989, Ausubel et al , 1992, Harlow et al 1989 (which
are incorporated herein by reference)
A "recombinant viral genome" comprises all or a part of a viral
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 In one embodiment, the transgene is flanked by flanking
elements The recombinant viral genome of the invention is embedded in the
genome of the carrier vector, and is ultimately packaged into a recombinant virus
A "recombinant virus" is a virus derived from the recombinant viral
genome described above The recombinant virus may comprise a transgene, may be an attenuated, replication-competent virus without a transgene, may be a
replication-competent virus with one or more point mutation(s), or may be a replication-deficient virus with one or more point mutations or genomic deletions,
or combinations thereof The recombinant virus comprising a transgene is capable
of transducing mammalian cells and delivering the transgene thereto
A "flanking element" or "flanking nucleic acid" is a nucleic acid
sequence generally derived from a mammalian virus which, when located in positions flanking a transgene, permits the packaging of the transgene into a
recombinant virus. Flanking elements may be the naturally-occurring flanking
elements from a mammalian virus which permit the packaging of the recombinant
virus, or may be artificial nucleic acid elements, e.g. mutated sequences of flanking
elements, that have the same or similar packaging function. Flanking elements
include, without limitation, the inverted terminal repeats (ITRs) of AAV or Ad, the long terminal repeats (LTRs) of retrovirus, the "α" or packaging sequence of herpes
simplex virus (HSV), as well as any other sequences that are required for packaging
from other viruses known in the art.
A "transgene" is a nucleic acid sequence that is to be delivered or
transferred to a mammalian cell. A transgene may encode a protein, peptide or polypeptide that is useful as a marker, reporter or therapeutic molecule. A
transgene may also encode a protein, polypeptide or peptide that is useful for
protein production, diagnostic assays or for any transient or stable gene transfer in
vitro or in vivo. Alternatively, a transgene may not encode a protein but rather be
used as an antisense molecule, ribozyme or other regulatory nucleic acid to inhibit
replication, transcription or translation of a nucleic acid to which it is
complementary or to target a complementary mRNA for degradation.
"Expression control sequences" are nucleic acid sequences that regulate the expression of a gene by being operably linked to the gene of interest.
"Operably linked" sequences include both expression control sequences that are
contiguous with the gene of interest and expression control sequences that act in
trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer
sequences; efficient RNA processing signals such as splicing and polyadenylation
signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance
translation efficiency (i.e., Kozak consensus sequence); sequences that enhance
protein stability; and when desired, sequences that enhance protein secretion.
As used herein, a "carrier vector" means a nucleic acid molecule
comprising a nonmammalian viral nucleic acid backbone and nucleic acid sequences
derived from mammalian sources, mammalian viral sources, nonmammalian sources,
and nonmammalian viral sources. The nonmammalian viral nucleic acid backbone
may be selected from a wide variety of sources, see, for example Table 1 of U.S. Pat. No. 5,731,182, herein incorporated by reference. The nonmammalian viral
nucleic acid backbone, upon transfection of the carrier vector nucleic acid into non¬
mammalian cells, is sufficient to produce packaged carrier virus comprising the
nucleic acid sequences inserted into the carrier vector.
A "carrier virus" is an encapsidated carrier vector capable of binding
to a mammalian cell and delivering the carrier vector's genome to the cell's nucleus.
As used herein, "ligand nucleic acid" means a nucleic acid which
encodes a protein which allows the carrier virus of the invention to bind to and
enter a mammalian cell. The nucleic acid encoding the protein may be operably
linked to expression control sequences that regulate the expression of the nucleic acid encoding the ligand.
"Helper function nucleic acid" is one or more nucleic acid sequences
that encode one or more proteins, peptides or polypeptides, or that is transcribed to
an RNA, wherein the one or more proteins, peptides, polypeptides or RNAs are
required by certain viruses for production of recombinant viruses. The sequences
may be naturally-occurring helper functions or may be sequences that have been
mutated or altered but which retain their respective helper functions. The sequences
may be derived from helper viruses or may be naturally-occurring or artificial
nucleic acid sequences that encode non-viral proteins that act as helper functions for
production of recombinant viruses. The nucleic acid sequences that are transcribed
to RNA or which encode the proteins, polypeptides or peptides may be operably linked to expression control sequences that regulate the expression of the nucleic
acid encoding the helper functions.
"Replication and/or encapsidation nucleic acid" is a nucleic acid
sequence or sequences which encode proteins or polypeptides that are required for replication and encapsidation of the recombinant virus. The sequences may be
naturally-occurring replication or encapsidation sequences or may be sequences that
have been mutated or altered but which retain their respective functions of
replication or encapsidation. The nucleic acid sequences encoding the proteins may
be operably linked to expression control sequences that regulate the expression of the nucleic acid encoding the replication and encapsidation sequences. A "replicon" is an episomal replication origin and those necessary
proteins (or DNA encoding these proteins) to initiate nucleic acid replication.
The Carrier Vector
The carrier vector of the invention is a chimeric vector backbone
derived from the nucleic acid of a nonmammalian virus. The carrier vector
comprises sufficient vector sequences to be able to replicate and encapsidate within
the appropriate nonmammalian host cell. The carrier vector also includes one or
more of the following inserts' an embedded recombinant viral genome, a ligand
nucleic acid providing for expression of a protein which can interact with a mammalian cell, replication and/or encapsidation nucleic acid required to replicate
and encapsidate a recombinant virus; and helper virus functions nucleic acids.
In a preferred embodiment, the carrier vector comprises an
embedded recombinant viral genome within its nonmammalian virus genomic
backbone The recombinant viral genome may comprise a transgene with associated expression regulatory sequences, wherein the transgene and regulatory
sequences are bordered by flanking elements of a mammalian virus Alternatively,
the recombinant viral genome does not contain a transgene but rather contains
deletions or point mutations in its sequence such that it produces an attenuated,
replication-proficient recombinant virus, or other deletions or point mutations that
produce a replication-deficient recombinant virus.
In a more preferred embodiment, the carrier vector comprises the
embedded recombinant viral genome and either or both of 1) nucleic acid sequences
encoding replication and/or encapsidation and 2) nucleic acid sequences encoding helper functions In an even more preferred embodiment of this invention, the
carrier vector additionally comprises a ligand nucleic acid providing for expression
of a protein which can interact with a mammalian cell In another preferred
embodiment, the ligand nucleic acid encodes a protein which can bind to a specific mammalian cell receptor
In the most preferred embodiment, the carrier vector comprises the
embedded recombinant viral genome and all of those nucleic acid inserts required
for production of a recombinant virus in a mammalian cell For instance, if the
carrier virus comprising the carrier vector is to be used to infect a cell line which expresses replication and encapsidation proteins for a recombinant AAV virus (e g ,
the A64 cell line described in U S Pat No 5,658,785 and the B50 cell line
described in PCT US98/19463), then the carrier vector would comprise the
embedded recombinant viral genome and the helper functions, and optionally the ligand nucleic acid Alternatively, if carrier virus is to be used to infect a cell line
which expresses a helper function for a recombinant AAV virus (e g , the 293 cell
line which expresses El), then the earner vector would comprise the embedded
recombinant viral genome, the replication and encapsidation nucleic acids for AAV
(/ ep and cap), and the helper functions required in addition to El (e g , E2a, E4ORF6 and VAI RNA), and optionally the ligand nucleic acid
If the earner virus is to be used to produce a recombinant retrovirus,
which does not require helper functions, the carrier vector would comprise the
embedded recombinant retroviral viral genome and the nucleic acids required for its replication and encapsidation (e g , gag, pol and env) and optionally, in cases where
the retrovirus is a lentivirus, one or more of the nucleic acids encoding regulatory or
auxilliary proteins (e g , tat, rev, ne vpr, vpu) If the earner virus is to be used to
produce a recombinant retrovirus in a cell line that expresses gag, pol and env or
the other functions described above in the case of lentiviruses, then the carrier virus
would need only comprise the embedded recombinant retroviral genome and
optionally the ligand nucleic acid Similarly, if the carrier virus is to be used to
produce a recombinant adenovirus, the earner vector would comprise the embedded
recombinant adenoviral genome and the nucleic acid sequences required for its replication and encapsidation The type of nucleic acid sequences required for
replication and encapsidation of the recombinant adenoviral genome depends upon
which adenoviral genes are deleted from the recombinant adenoviral genome and
whether the mammalian cell line that the earner virus infects expresses any adenoviral genes (e g , 293 cells express El) Any carrier vector genome may
optionally comprise a ligand nucleic acid to increase infection by the carrier virus of
a mammalian cell
The embedded recombinant viral genome and other nucleic acid
inserts may be carried on separate carrier vectors, but in the most preferred embodiment, the embedded recombinant viral genome and all other desired nucleic
acid inserts are carried on a single carrier vector The advantage of a single carrier
vector is that only a single infection by the earner virus of the mammalian host cell
is required in order to produce a recombinant virus
In another embodiment of the invention, the inability of the carrier vector to replicate in mammalian cells is overcome by supplying a mammalian
rephcon to the carrier vector The provision of a replicon assures that mammalian
cells infected by the carrier vector maintain a sufficient copy number of the carrier
vector extrachromosomally throughout a population of proliferating and dividing
mammalian cells
Based on this description, other embodiments of the carrier vector
will be readily apparent to those of ordinary skill in the art
Nonmammalian Virus Backbone
The chimeric carrier vector is constructed from a backbone of a
nonmammalian virus The backbone need not be the entire genome of the nonmammalian virus, but may be only that portion of the genome necessary for
replication in a nonmammalian host Preferably, the vector backbone is derived
from an invertebrate virus Table 1 of U S Pat No 5,731,182 lists several
examples of viruses that may be used to form the backbone of the chimeric vector,
the sequences of which are available from various sources, such as Genbank In a
preferred embodiment, the invertebrate DNA virus is a baculovirus In a more
preferred embodiment, the bacuolovirus is a Granulovirus or Nucleopolyhedrovirus
In an even more preferred embodiment, the nonmammalian viral backbone is
derived from the baculovirus Autographa californica nuclear polyhedrosis virus
(AcNPV) See, e g , GenBank Accession No L22858
In a preferred embodiment, the nonmammalian virus backbone must
be capable of replication in its ordinary host cell, but incapable of replication in a
mammalian cell For example, the baculovirus virus backbone exemplified herein replicates only in insect cells
The Embedded Recombinant Viral Genome
The methods of the present invention allow for large scale
production of high titers of recombinant virus, i.e., one that has a transgene inserted
therein to be delivered to target mammalian cells, or one that does not have a
transgene but rather has a mutation or deletion in a viral gene and is to be used as a
vaccine, e.g., an attenuated and replication-proficient recombinant virus or a
replication-deficient mutant virus. The recombinant virus may be any virus of
interest for use to deliver transgenes to mammalian cells or for use as a vaccine.
Preferred recombinant viruses for delivery of a transgene include adenoviruses,
retroviruses, adeno-associated viruses, herpesvirus amplicons and hepatitis B
viruses.
In order to manufacture a recombinant virus containing a transgene,
the method of the present invention 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 the packaging elements of the virus to be manufactured, in place of the
genes normally found therein. Where the length of the replacement is shorter than
that being replaced, and that shorter length would pose an obstacle to proper
packaging, an optional spacer or "stuffer" sequence may be inserted in order to
maintain the proper length for packaging. The entire construct of the ERS- transgene constructed bordered by the flanking elements is the genome of the
recombinant virus of the present invention, which is then embedded in the carrier vector's genome, at which point it subsists as an embedded recombinant viral
genome. Each of these elements is described in detail below:
'The Transgene
The composition of the transgene sequence depends upon the
intended use for the resulting recombinant virus. For example, one type of
transgene sequence comprises a reporter or marker sequence, which upon
expression produces a detectable signal. Such reporter or marker sequences
include, without limitation, DNA sequences encoding E. coli β-lactamase, β-
galactosidase (LacZ), alkaline phosphatase, HSV thymidine kinase, green
fluorescent protein (GFP), bacterial chloramphenicol acetyltransferase (CAT),
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.
These 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. For example, where the transgene is the LacZ
gene, the presence of a recombinant virus is detected by assays for β-galactosidase activity. Similarly, where the transgene is luciferase, the recombinant virus gene
expression may be measured by light production in a luminometer.
However, desirably, the transgene is a non-marker gene which can
be delivered to a cell or an animal via the recombinant virus produced by this
method. The transgene may be selected from a wide variety of gene products useful
in biology and medicine, such as proteins, antisense nucleic acids (e.g., RNAs), or
catalytic RNAs. The invention may be used to correct or ameliorate gene
deficiencies, wherein normal genes are expressed but at less than normal levels, and
may also be used to correct or ameliorate genetic defects wherein a functional gene
product is not expressed. A preferred type of transgene sequence is a therapeutic gene which expresses a desired corrective gene product in a host cell. These
therapeutic nucleic acid sequences typically encode products which, upon
expression, are able to correct, complement or compensate an inherited or non-
inherited genetic defect, or treat an epigenetic disorder or disease. However, the
selected transgene may encode any product desirable for study. The selection of the transgene sequence is not a limitation of this invention. Choice of a transgene
sequence is within the skill of the artisan in accordance with the teachings of this
application.
The invention also includes methods of producing recombinant virus
and compositions thereof which can be used to correct or ameliorate a gene defect
caused by a multi-subunit protein. In certain situations, a different transgene may be used to encode each subunit of the protein. This may be desirable when the size
of the 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 recombinant virus expressing
each of the different subunits
Alternatively and more preferably, different subunits of a protein
may be encoded by the same transgene In this case, a single transgene would
include the DNA encoding each of the subunits, with the DNA for each subunit
separated by an internal ribosome entry site (IRES) The use of IRES permits the
creation of multigene or polycistronic mRNAs IRES elements are able to bypass
the nbosome scanning model of 5' methylated cap-dependent translation and begin
translation at internal sites (Pelletier and Sonenberg, 1988) IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have
been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian
mRNA (Macejak and Sarnow, 1991) IRES elements can be linked to heterologous
open reading frames By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation Thus, multiple genes can be
efficiently expressed using a single promoter/enhancer to transcribe a single
message This is preferred when the size of the DNA encoding each of the subunits
is sufficiently small that the total of the DNA encoding the subunits and the IRES is
no greater than the maximum size of the DNA insert that the virus can encompass For instance, for rAAV, 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, β-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), bFGF2, acidic
fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming
growth factor (TGFα), platelet-derived growth factor (PDGF), insulin-like
growth factors I and II (IGF-I and IGF-II), any one of the transforming growth
factor β (TGFβ) superfamily comprising TGFβ, activins, inhibins, or any of the
bone morphogenic proteins (BMP) BMPs 1-15, any one of the
heregulin/neuregulin ARIA/neu differentiation factor (NDF) family of growth
factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3, NT-4/5 and NT-6, ciliary neurotrophic factor (CNTF), glial
cell line derived neurotrophic factor (GDNF), neurtuin, persephin, agrin, any one of
the family of semaphorins/collapsins, netrin-1 and netrin-2, hepatocyte growth
factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase.
Other useful gene products include proteins that regulate the immune system including, without limitation, cytokines and lymphokines such as thrombopoietin (TPO), interleukins (IL) IL-lα, IL-lβ, TL-2, LL-3, IL-4, IL-5, IL-6,
IL-J IL-8, IL-9, IL-10, ΓL-11, IL-12, LL-13, IL-14, IL-15, IL-16, and IL-17,
monocyte chemoattractant protein (MCP-1), leukemia inhibitory factor (LIF), granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony
stimulating factor (G-CSF), monocyte colony stimulating factor (M-CSF), Fas
ligand, tumor necrosis factors and β (TNFα and TNFβ), interferons (IFN) IFN- ,
IFN-β and IFN-γ, stem cell factor, fTk-2/flt3 ligand. Gene products produced by the 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,
chimeric T cell receptors, single chain T cell receptors, class I and class II MHC
molecules, as well as engineered MHC molecules including single chain MHC molecules. Useful gene products also include complement regulatory proteins such
as membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, CR2
and CD59.
Still other useful gene products include any one of the receptors for
the hormones, growth factors, cytokines, lymphokines, regulatory proteins and immune system proteins. Examples of such receptors include flt-1, flk-1, TIE-2,
the trb family of receptors such as TrkA, MuSK, Eph, PDGF receptor, EGF
receptor, HER2, insulin receptor, IGF-1 receptor, the FGF family of receptors, the
TGFβ receptors, the interleukin receptors, the interferon receptors, serotonin
receptors, α-adrenergic receptors, β-adrenergic receptors, the GDNF receptor, p75
neurotrophin receptor, among others. 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
selectins E-selectin, P-selectin and L-selectin. The invention encompasses receptors
for cholesterol regulation, including the LDL receptor, HDL receptor, VLDL
receptor, and the scavenger receptor. 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
superfamily including glucocorticoid receptors and estrogen receptors, Vitamin D
receptors and other nuclear receptors. In addition, useful gene products include
antimicrobial peptides such as defensins and maginins, transcription factors such as juif f s, max, mad, serum response factor (SRF), AP-1, AP-2, myb, MRG1,
CREM, Alx4, FREAC1, NF-κB, members of the leucine zipper family, C2H4 zinc
finger proteins, including Zif268, EGR1, EGR2, C6 zinc finger proteins, including
the glucocorticoid and estrogen receptors, POU domain proteins, exemplified by
Pitl, homeodomain proteins, including HOX-1, basic helix-loop-helix proteins, including myc, MyoD and myogenin, ETS-box containing proteins, TFE3, E2F,
ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SPI, CCAAT-
box binding proteins, interferon regulation factor 1 (IRF-1), Wilms tumor protein,
ETS-binding protein, STAT, GATA-box binding proteins, e.g., GATA-3, and the forkhead family of winged helix proteins.
Other useful gene products include carbamoyl synthetase I, ornithine
transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase,
fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha- 1 antitrypsin,
glucose-6-phosphatase, porphobilinogen deaminase, factor VII, factor VIII, factor
IX, factor II, factor V, factor X, factor XII, factor XI, von Willebrand factor,
superoxide dismutase, glutathione peroxidase and reductase, heme oxygenase, 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 transmembrane regulator (CFTR), the product of Wilson's disease gene
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, αl 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.
Other useful transgenes include non-naturally occurring
polypeptides, such as chimeric or hybrid polypeptides or polypeptides having a non- naturally occurring amino acid sequence containing insertions, deletions or amino acid substitutions. For example, single-chain engineered immunoglobulins could be
useful in certain immunocompromised patients Other useful proteins include
truncated receptors which lack their transmembrane and cytoplasmic domain
These truncated receptors can be used to antagonize the function of their respective
ligands by binding to them without concomitant signaling by the receptor Other
types of non-naturally occurring gene sequences include antisense molecules and
catalytic nucleic acids, such as ribozymes, which could be used to reduce
overexpression of a gene
Other useful transgenes include those that encode antigemc peptides
capable of generating an immune response Recombinant vectors comprising these
transgenes can be used for genetic immunization Useful transgenes include those
that encode peptides specific for Epstein Barr virus, HIV, simian immunodeficiency
virus (SIV), human T-cell leukemia viruses I and II (HTLV-I and HTLV-II),
hepatitis A, B, C, D and E, pseudorabies virus, rabies virus, cytomegalovirus,
respiratory syncytial virus, parainfluenza virus types 1-4, mumps virus, rubella virus,
polio virus, rubeola virus, influenza virus types A, B and C, rotavirus, herpes
simplex viruses types 1 and 2, varicella-zoster virus, human herpes virus type 6,
hantavirus, adenoviruses, chlamydia pneumoniae, chlamydia trachomatis,
mycoplasma pneumoniae, mycobacterium tuberculosis, atypical mycobacteria, feline
leukemia virus, feline immunodeficiency virus, bovine immunodeficiency virus,
equine infectious anemia virus, caprine arthritis encephalitis virus, visna virus, Staphlococcus species and Streptococcus species The transgenes may also be directed against peptides from tumor antigens to provide immunization for tumors
and cancers
Expression Control Sequences
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
encoding the replication and encapsidation functions of the recombinant virus, the
helper functions and the ligand. The choice of expression control sequence depends
upon the type of expression desired. For eukaryotic cells, expression control
sequences typically include a promoter, an enhancer, such as one derived from an immunoglobulin gene, SV40, cytomegalovirus, etc., and a polyadenylation sequence which may include splice donor and acceptor sites. 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 SV-40, and is referred to as the SV-40 T intron sequence.
Another vector element that may be used is an internal ribosome entry site (IRES),
as described above. An IRES sequence is used to produce more than one polypeptide from a single gene transcript. An IRES sequence 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, John Wiley & Sons, New York, 1989]
In one embodiment, high-level constitutive expression will be desired Examples of such promoters 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
(1 85)], the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic
β-actin promoter and the phosphoglycerol kinase (PGK) promoter
In another embodiment, 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
sheep 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
system [Gossen et al , Science, 268: 1766-1769 (1995); see also Harvey et al., Curr
Opin Chem Biol . 2.512-518 (1998)]; the RU486-inducible system [Wang et al.,
Nat Biotech . 15.239-243 (1997) and Wang et al , Gene Ther . 4.432-441 (1997)];
and the rapamycin-inducible system [Magari et al., J. Clin Invest . 100.2865-2872
(1997), Rivera et al . Nat Medicine. 2: 1028-1032 (1996)]. Other types of inducible
promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, or in replicating cells
only In a preferred embodiment, the transgene is under the control of the native p5
promoter of AAV
In another embodiment, the native promoter for the transgene or
nucleic acid sequence of interest will be used The native promoter may be
preferred when it is desired that expression of the transgene or the nucleic acid
sequence should mimic the native expression The native promoter may be used
when expression of the transgene or other nucleic acid sequence must be regulated
temporally or developmentally, or in a tissue-specific manner, or in response to
specific transcπptional stimuli In a further embodiment, other native expression
control elements, such as enhancer elements, polyadenylation sites or Kozak
consensus sequences may also be used to mimic the native expression
In one embodiment, 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 α-actin, myosin light chain 2A,
dystrophin, muscle creatme kinase, as well as synthetic muscle promoters with
activities higher than naturally-occurring promoters [see Li et al , Nat Biotech .
17 241-245 (1999)] Examples of promoters that are tissue-specific are known for
liver [albumin, Miyatake et al J Virol , 71 5124-32 (1997), hepatitis B virus core
promoter, Sandig et al , Gene Ther , 3 1002-9 (1996), alpha-fetoprotein (AFP),
Arbuthnot et al , Hum Gene Ther . 7 1503-14 (1996)], bone [osteocalcm, Stein et al , Mol Biol Rep . 24 185-96 (1997), bone sialoprotein, Chen et al , J Bone Miner Res . 11 654-64 (1996)], lymphocytes [CD2, Hansal et al , J Immunol .
161 1063-8 (1998), lmmunoglobu n heavy chain, T cell receptor a chain], neuronal
[neuron-specific enolase (NSE) promoter, Andersen et al. Cell Mol Neurobiol ,
13 503-15 (1993), neurofilament light-chain gene, Piccioli et al , Proc Natl Acad
Sci USA, 88 561 1-5 (1991), the neuron-specific vgf gene, Piccioli et al , Neuron,
15 373-84 (1995)], among others.
Of course, not all vectors and expression control sequences will
function equally well to express all of the transgenes or other nucleic acid sequences
of this invention However, one of skill in the art may make a selection among
these expression control sequences without departing from the scope of this
invention Suitable promoter/enhancer sequences which function in the appropriate
host cell of choice may be selected by one of skill in the art using the guidance
provided by this application. Such selection is a routine matter and is not a
limitation of the molecule or construct.
In one method of identifying a suitable expression control sequence
for a desired nucleic acid sequence, one may select one or more expression control
sequences and operably link the expression control sequence to the nucleic acid sequence to be regulated. Then, one may insert these operably linked sequences
comprising the expression control sequence and regulated sequence into the
genome of the carrier vector. In one embodiment, one may insert a recombinant viral genome comprising the expression control sequence and the transgene into a
nonmammalian vector of the instant invention After following one of the methods for producing and packaging the recombinant vector as taught in this specification
one may infect suitable cells in vitro or in vivo. The number of copies of the
transgene in the cell may be monitored by Southern blotting or quantitative PCR,
the level of RNA expression may be monitored by Northern blotting or quantitative
RT-PCR, and the level of protein 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
In a similar fashion, one may select one or more expression control
sequences and operably link it to a nucleic acid sequence encoding replication and
encapsidation proteins, helper functions or a ligand, and insert the resultant desired
nucleic acid molecule into a vector of the instant invention. One may also select one or more vector replication sequences and insert them into a vector of the instant
invention After packaging and infecting nonmammalian cells, one may measure the
particular effects, e g , on expression of the ligand or on replication of the vector,
by one of the methods described above. One may also use a functional test to
determine if one or more particular expression control sequences operably linked to
a nucleic acid sequence encoding a ligand produces a carrier virus which is able to
infect mammalian cells efficiently. One may assay a number of different expression
control sequences to determine which one is most effective for mammalian cell
infection. The same may be done using a variety of vector replication sequences.
Furthermore, after infecting mammalian host cells and obtaining
recombinant virus, one may infect mammalian cells with the recombinant virus, then
measure the expression of the replication and encapsidation proteins and/or helper functions by one of the methods described above. One may also use a functional
test to determine if one or more particular expression control sequences operably
linked to one or more helper functions or replication or encapsidation functions is
capable of supporting production of a infectious recombinant virus One may
determine which of many expression control sequences are most effective in
producing a high titer of infectious recombinant virus
Flanking Elements
Flanking elements are required for replication, excision and
packaging of many viruses, and each type of virus has its own type of flanking
elements In a wild-type virus, these elements flank the viral genes when the viral
DNA integrates into a host cell chromosome In the case of integrating viruses,
when the wild-type virus is rescued from the host chromosome, the flanking
elements excise along with the viral DNA and remain in flanking positions surrounding the rescued viral DNA, in a form suitable for packaging into virions
For non-integrating, extrachromosomal viruses (e g HSV), flanking sequences
serve functions in DNA replication and packaging In recombinant viruses, much or
all of the viral nucleic acid sequences between the flanking elements are removed
from the virus and are replaced with a transgene and its associated expression
regulatory sequences
In one embodiment of the invention, the recombinant virus is a
recombinant adenovirus, and comprises a selected transgene operably linked to
expression regulatory sequences and the adenoviral flanking elements Adenoviral flanking elements are ITRs and are 100-200 bp in length A large number of
adenoviral flanking elements are known, such as those from 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]
In another embodiment, the recombinant virus is a recombinant
retrovirus, and comprises a selected transgene operably linked to expression
regulatory sequences and retroviral flanking elements The flanking elements are
long terminal repeat (LTR) sequences that are present at the 5' and 3' ends of the
retroviral genome These LTRs contain strong promoter and enhancer sequences
and are also required for integration in the host cell genome (Coffin, 1990) A large number of retroviral LTRs are known. See, for instance, U S Pat No 5,672,510
In yet another embodiment of the invention, the recombinant virus is
a recombinant AAV, and comprises a selected transgene operably linked to expression regulatory sequences and AAV flanking elements The naturally-
occurring AAV ITRs consist of approximately 145 bp at the 5' and 3' ends of the
AAV genome The AAV ITRs are required for replication, excision and encapsidation of both wild type and recombinant AAV virions
In another embodiment, the recombinant virus is either a herpesvirus
derivative containing one or more mutations or deletions of viral genes, or is a
herpesvirus amplicon In either case, the flanking elements would be the viral
terminal repeats (e g , the "α " sequence if the virus is HSV) HSV amplicons are defective HSV genomes containing the packaging sequence ( ), viral origin of DNA
replication (on) and the transgene cassette of interest operably linked to the desired
expression regulatory sequences In the presence of helper herpesvirus or substitute
helper functions, the amplicon is replicated and packaged as head-to-tail
concatemers to form wild-type size genomes.
In another embodiment, the recombinant virus is a recombinant
defective hepatitis B virus (HBV) and comprises a selected transgene operably
linked to expression regulatory sequences and inserted into the HBV genome. In
vitro studies have shown that the recombinant hepatitis B virus retains the ability for
helper-dependent packaging and reverse transcription despite the deletion of up to
80% of its genome (Horwich et al., 1990). This suggests that large portions of the genome may be replaced with foreign genetic material. The hepatotropism and
persistence (integration) were particularly attractive properties for liver-directed
gene transfer.
Liuand DNA
Most nonmammalian viruses are not infectious to mammalian cells;
however, it has been reported that in some cases, nonmammalian viruses will infect
certain particularly infection-susceptible mammalian cell lines. [Barsoum et al.,
Human Gene Therapy 8:2011-2018 (Nov. 20, 1997)]. Where the host cell to be
used for manufacturing the recombinant virus is not susceptible to infection by the
nonmammalian virus, the nonmammalian virus may be modified by incorporating
ligand DNA in the nonmammalian backbone. The nonmammalian backbone may
also be modified by incorporation of ligand DNA to increase infection of mammalian host cell by the nonmammalian virus. The expression of the ligand
DNA by the subsequently produced nonmammalian virus will permit infection or
mcrease infection of mammalian cells The backbone of the carrier vector of the
present invention is modified by addition of DNA encoding components needed to
produce a ligand which is recognized by a desired mammalian cell
The ligand DNA is selected from genes which, when expressed, yield
a gene product that will be present on the surface of the encapsidated carrier vector,
thus presenting the ligand to the mammalian cell target receptors and allowing the
carrier vector to bind to and enter mammalian cells [Barsoum et al , Human Gene
Therapy 8 201 1-2018 (Nov 20, 1997) ] The ligand DNA is placed under the
regulatory control of expression regulatory sequences that insure that the ligand
gene product is expressed coordinately with the replicated DNA of the carrier
vector to allow efficient chimeric carrier vector production in nonmammalian host
cells The nonmammalian expression regulatory sequence may be identical, similar,
or distinct from the backbone's native regulatory sequences, so long as it is capable
of regulatory functions in the nonmammalian host cell In a preferred embodiment,
the expression regulatory sequences comprise a promoter derived from the native
nonmammalian virus from which the nonmammalian vector backbone is derived In
a more preferred embodiment, the promoter is polyhedπn early promoter (polH)
and the nonmammalian vector backbone is derived from baculovirus
In general, if a ligand DNA is regulated by nonmammalian
expression regulatory sequences, the ligand DNA will not be expressed when the
chimeric vector infects the mammalian host cell The absence of expression of the
ligand encoded by the DNA may be useful to prevent incorporation of ligand mto
the recombinant virus coat encoded from the carrier nonmammalian vector, which
could disrupt its structural integrity or cause adverse immunogemc reactions in an animal However, if ligand expression is desired in the mammalian host cell, then
alternative or additional expression regulatory sequences may be operably linked to
the sequences encoding the ligand to permits its expression in mammalian and
nonmammalian host cells Alternatively, there may be some instances in which the
ligand DNA is expressed because the nonmammalian expression regulatory
sequences are also activated in the mammalian cells
The ligand DNA can be essentially any nucleic acid that encodes a
protein, polypeptide or peptide that modifies the mature nonmammalian virus to
enable it to bind to and enter mammalian cells The ligand can be naturally- occurring protein, a fragment of a naturally-occurring protein that has a desired
binding capability, or an artificial or mutated polypeptide or peptide that has a
desired binding capability The ligand can be one of general specificity, which
would allow binding to a wide variety of mammalian cells (e g , vesicular stomatitis
virus glycoprotem G (VSV-G) gene, bovine syncytial virus (BSV) envelope
glycoprotem gene, or amphotropic envelope gene as illustrated below), or it may be
more specific, allowing binding to targeted specific cell types For instance, the
ligand may cause the virus to bind via electrostatic interactions or other general
mechanism of interacting the mammalian cell, or it may be a specific ligand-receptor interaction
Useful ligand nucleic acids may be any nucleic acid which encodes a ligand that permits the nonmammalian virus to interact with the mammalian cell
For instance, the ligand may be one which increases the electrostatic interaction
between the virus and the mammalian cell for a receptor found on the mammalian
host cells that are to be infected by the carrier virus Other useful ligand nucleic
acids include, without limitation, nucleic acids encoding peptide hormones, growth
factors, or other normally secreted factors for which the mammalian host cell of
interest expresses a receptor The nucleic acids useful as a ligand include all those
secreted factors, peptide hormones and growth factors which have a normal cellular
receptor and which are disclosed above for transgenes For instance, the ligand
nucleic acid may encode PDGF, EGF, bFGF, aFGF, insulin, IGF-I, IGF-II, apoE,
apoAl, apoA4, EPO, PTH, GH or GRF The ligand nucleic acid may encode a
native or genetically engineered immunoglobulin (e g , ScFv, chimeric immunoglobulin, humanized immunoglobulin, etc ) or MHC molecule that
specifically binds to a particular cell surface protein on the mammalian cell Other
ligand nucleic acids of interest encode a member of the extracellular matrix such as
a collagen, elastin, thrombospondin, tenascin or vitronectin, which bind to integrins
and other cellular transmembrane receptors The nucleic acid sequence encoding a
ligand which is normally secreted may be modified by incorporating a nucleic acid
sequence encoding an "anchoring domain" at either the 5' or 3' end of the coding
sequence for the ligand The anchoring domain is a region that secures the ligand in
the viral coat In a preferred embodiment, the anchoring domain is at the 3' end of
the coding sequence for the ligand In a further preferred embodiment, the
anchoring domain is derived from a viral coat protein, such as HIV gp41 (which
anchors gpl20 coat protein to the viral envelope) Other examples include E protein of dengue virus or the 14 kDa protein of vaccinia virus
The ligand nucleic acid also may encode a protein that is normally
anchored in the cell membrane of a mammalian cell which binds to a particular cell
surface protein or counter-receptor on a mammalian host cell Examples of this
type of ligand nucleic acid include a number of the CD antigens, such as the T cell
receptor (TCR), CTLA-4 receptor and B-7, integrins such as Mac-1, LFA-1, and
pi 50,95, intercellular adhesion molecules such as ICAM-1, ICAM-2, ICAM-3 and
ICAM-4, and selectins, such as E-selectin, P-selectin and L-selectin The ligand may also be an artificial or mutated counter-receptor, such as a cell-surface
anchored or hybrid immunoglobulin or TCR.
In one embodiment, the ligand is one that is normally present on a
virus and which mediates binding to a mammalian cell, for example, gpl20 of HIV
or HA from influenza In another embodiment, the ligand is one that is normally
present on a bacterial cell and which mediates binding to a mammalian cell, for example, Protein A from Staphylococcus aureus is known to bind to
immunoglobulins.
In another embodiment, the mammalian host cell is genetically
engineered to express a receptor which specifically binds to a ligand. Thus, one can
design mammalian host cell-carrier virus systems that promote highly specific
binding of the carrier virus to the mammalian host cell. For example, one may
engineer a mammalian host cell line to express a growth factor receptor, such as the
EPO receptor, and design the carrier vector to comprise a ligand nucleic acid
comprising the EPO gene. One of skill in the art, in light of the instant specification, would be able to identify a large number of mammalian host cell-
carrier virus interactive receptor-ligand systems.
In one embodiment the ligand DNA is the VSV-G gene This gene may be placed under the control of the baculovirus polyhedrin (pPH) early
promoter The VSV-G protein, when expressed, modifies the mature carrier virus
such that it may bind to mammalian host cells and thereby infect them [Barsoum,
supra] In another embodiment of the present invention, the ligand DNA is the
BSV env gene, which functions in the context of the invention in a similar manner.
In another preferred embodiment, the present invention exploits the
fact that nonmammalian viruses normally do not terminate glycoproteins with sialic
acid Thus, the ligand DNA is a gene which expresses an asialoglycoprotein, which
binds to mammalian lectins (e.g., the hepatic asialoglycoprotein receptor), which
would then facilitate entry into the mammalian cell
Replication and Encapsidation Nucleic Acids
The replication and encapsidation functions are required for
replication, excision and encapsidation of the recombinant viral genome into an
infectious recombinant virion or virus. Each type of recombinant virus will require
a different type of replication and encapsidation function. For instance, if the
recombinant virus is a retrovirus, then the replication and encapsidation functions
include the retroviral gag, pol and env genes (and in the case of lentiviruses will also include regulatory or accessory genes such as HIV tat, rev, nef, vpu or vpr), while if the recombinant virus is an AAV, then the replication and encapsidation functions include the rep and cap genes from an AAV.
As discussed above, either the carrier vector or the mammalian host cell may comprise nucleic acids encoding those replication and encapsidation
functions required for a particular recombinant virus. Mammalian host cells such as
A64 cell line described in U.S. Pat. No. 5,658,785 and the B50 cell line described in
PCT US98/19463) express AAV rep and cap genes for replication and packaging
of recombinant AAV. Similarly, mammalian host cells expressing adenoviral genes
required for replication and packaging of recombinant adenovirus are known [see,
e.g., U.S. Pat. No. 5,851,806 and Amaltifano et al., Proc. Natl. Acad. Sci. USA
93 :3352-6 (1996)] or may be constructed, and a number of mammalian host cells expressing retroviral genes required for replication and packaging of recombinant
retroviruses have been constructed [see, e.g., Cone et al., Proc. Natl. Acad. Sci.
USA 81 :6349-6353 (1984); Miller et al., Mol. Cell. Biol. 6:2895-2902 (1986); Miller et al, Mol. Cell. Biol. 5:431-437 (1985); and Sorge et al., Mol. Cell. Biol.
4: 1730-1737 (1984)]. Cell lines comprising genes required for packaging of
herpesviruses (see, e.g., U.S. Pat. No. 5,851,826) are also known.
If a cell line comprises all the necessary replication and encapsidation
functions to replicate, excise and package a particular recombinant viral genome,
then the carrier vector need not comprise any replication and/or encapsidation
nucleic acid sequences. The cell line may comprise the necessary replication and
encapsidation functions either by being transiently or stably transduced with the
nucleic acid encoding the appropriate proteins. In a preferred embodiment, the cell
line stably comprises the replication and encapsidation functions. Furthermore, the
cell line may express the replication and encapsidation functions constitutively or
inducibly Constitutive or inducible expression may be controlled by using any of
the expression regulatory sequences known in the art or as discussed above under
"Expression Regulatory Sequences." In a preferred embodiment, the expression of
the replication and encapsidation functions is inducible. In a more preferred
embodiment, the replication and encapsidation functions are stably transfected or
infected and are inducibly expressed In an even more preferred embodiment, the
expression of the replication and encapsidation functions is regulated by their native
promoters
A mammalian cell line used in the instant invention may comprise none of the functions required for replication or encapsidation, or may comprise
only a part of the functions required for replication or encapsidation If a
mammalian cell line comprises none of the functions required for replication or
encapsidation, these functions must be introduced into the cell by a vector for production of the recombinant virus. In a preferred embodiment, one or more
carrier viruses of the instant invention are used to transduce the mammalian cell line
with the nucleic acids encoding the replication and encapsidation functions. In a
more preferred embodiment, a single carrier virus comprising the replication and
encapsidation functions are used to transduce the mammalian cell line In an even
more preferred embodiment, a single carrier virus comprising the replication and
encapsidation functions, the embedded recombinant viral genome, and any other
nucleic acid sequences required for recombinant virus production are used to transduce the mammalian cell line.
If the mammalian cell line comprises some of the replication or
encapsidation functions, these functions must be introduced into the cell by a vector
for production of the recombinant virus. In a preferred embodiment, one or more
carrier viruses are used to transduce the mammalian cell line with the nucleic acids
encoding the missing replication and encapsidation functions. In a more preferred
embodiment, a single carrier virus comprising the missing replication and
encapsidation functions are used to transduce the mammalian cell line. In an even more preferred embodiment, a single carrier virus comprising the missing replication
and encapsidation functions, the embedded recombinant viral genome, and any
other nucleic acid sequences required for recombinant virus production are used to
transduce the mammalian cell line.
The replication and encapsidation functions required for a
recombinant virus differ depending upon the type of recombinant virus. In general, the required replication and encapsidation functions are known in the art for the
various recombinant viruses. In preferred embodiment of recombinant vectors,
recombinant AAV requires rep and cap for replication and encapsidation,
recombinant retroviruses require gag,pol and env (and tat, rev and nef Tor
lentiviruses), recombinant adenoviruses require all of part of the functions encoded
by El, E2, E4, L1-L5, pIX and IVa2 genes, alone or in combination, and
recombinant herpesviruses require a large number of genes, which may be provided
by a helper herpesvirus or by a carrier vector comprising the required herpesvirus
genes. Together the host mammalian cell and the carrier virus must contribute the necessary replication and encapsidation functions for the particular recombinant virus in order to obtain infectious recombinant virus from the mammalian host cells.
In one embodiment, the replication and encapsidation functions are encoded by nucleic acids encoding the naturally-occurring proteins having the
replication and encapsidation functions In another embodiment, the replication and
encapsidation functions are encoded by nucleic acids encoding fragments or mutems
of the naturally-occurring proteins but which retain their respective replication and
encapsidation functions In another embodiment of the invention, other
recombinant viruses may be produced using nucleic acids encoding the appropriate
replication and encapsidation functions for the particular recombinant virus desired
Other types of recombinant viruses and the replication and encapsidation functions they require are known in the art
In a preferred embodiment, when production of a recombinant AAV
is desired, the rep and cap sequences are regulated by a native AAV p5 promoter
In another preferred embodiment, when production of a recombinant adenovirus is
desired, the nucleic acid sequences encoding the replication and encapsidation
functions for adenovirus are regulated by their native adenovirus promoters Native promoters may also be used for regulating the expression of replication and
encapsidation functions of other recombinant viruses, including, without limitation,
herpesvirus and HBV
In a more preferred embodiment, the replication and encapsidation
functions are encoded by nucleic acid sequences inserted in the carrier vector The advantage of having these sequences on the carrier vector is that no cell line has to
be constructed before infection by the carrier virus It is often difficult to create and
maintain cell lines expressing replication and encapsidation functions because many
of the proteins that provide these functions are toxic to mammalian cells. Thus,
another advantage of inserting the replication and encapsidation sequences on the
carrier vector is that the replication and encapsidation functions are only expressed
in the mammalian cells when the cells are infected with the carrier virus when the
production of a recombinant virus is desired. In a more preferred embodiment, the
carrier virus has an embedded recombinant viral genome comprising a transgene
and the ITRs from AAV and further has rep and cap gene sequences for replication
and encapsidation of the embedded recombinant AAV genome. In an even more
preferred embodiment, the expression of the rep and cap genes is regulated by their
native promoters or rep/cap is separated from the promoter to decrease or eliminate
homologous or non-homologous recombination to form wt AAV. Similarly, in a
preferred embodiment of carrier viruses that produce recombinant retrovirus,
adenovirus, herpesvirus and HBV, the carrier viruses contain nucleic acid sequences that encode replication and encapsidation functions. In a more preferred
embodiment, the nucleic acid sequences encoding the replication and encapsidation
functions are regulated by their native promoters.
Helper Functions
A number of viruses are unable to replicate, excise and package on their own, and require helper functions to do so. Helper functions may also be
required for the production of recombinant viruses which have had a large amount
of their genome deleted for insertion of the transgene. The nature of the helper function may differ depending upon the type of recombinant virus and/or the
amount of genome that has been deleted. Helper functions include viral proteins,
non-viral proteins, as well as physical and/or chemical agents. One may identify which helper functions are required from what is known in the art. For instance, it
is known that AAV requires helper functions from adenovirus or herpesvirus or
from different chemical or physical agents. Alternatively, one of skill in the art may
determine what helper functions are required by producing recombinant viruses
using the composition and methods disclosed in the instant specification
To identify which helper functions are required for high levels of recombinant virus production, one may infect mammalian host cells with the carrier
virus in the absence of helper functions and measure the titer of infectious
recombinant virus One may then transduce the mammalian host cells with various
nucleic acids encoding potential helper functions. Such helper functions may be any nucleic acid that is known or thought to encode a helper function. In a preferred
embodiment, the helper function is one or more viral proteins. In a more preferred
embodiment, the helper virus proteins are insufficient to produce a mature helper
virus After transducing the mammalian host cell with the nucleic acid encoding the
potential helper function, one may then measure the titer of the recombinant virus.
If the carrier virus comprises a recombinant AAV genome, helper
functions are required for production of infectious recombinant AAV In a
preferred embodiment, the helper functions are nucleic acids derived from a virus
In a more preferred embodiment, the helper functions are derived from adenovirus,
herpes simplex virus (HSV) HSV-1, HSV-2, cytomegalovirus (CMV) or pseudorabies virus (PRV). In an even more preferred embodiment, the helper
functions are at least El a, Elb and E2a from adenovirus, and may also include E4ORF6 and VAI In another preferred embodiment, 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) The helper
functions may also include all 7 HSV DNA replication genes (UL5, 8, 52, 29, 30, 9
and 42) Alternatively, 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 recombinant virus
may be delivered to the mammalian 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 Alternatively, the mammalian cell line may stably express (either on an
extrachromosomal episome or through integration in the cell's genome) the helper functions In addition, some of the helper functions may be expressed by the
mammalian cell line while other helper functions are introduced by a vector. For
example, 293 cells (ATCC CRL-1573) constitutively produce adenoviral El a and
Elb proteins. Thus, for production of recombinant AAV, the helper functions required for the production of infectious recombinant AAV, such as E2A, E4ORF6 and VAI, are introduced into the host cell by transfection or infection of a vector.
In a preferred embodiment, the helper functions are transduced into
the mammalian cells by a carrier virus. In a more preferred embodiment, some or all of the helper functions are transduced into the mammalian cell by a carrier virus
comprising the embedded recombinant viral genome. In an even more preferred
embodiment, all of the helper functions are transduced into the mammalian cell by a carrier virus comprising the embedded recombinant viral genome, any required
replication and encapsidation functions, and, optionally, a ligand DNA. In the most
preferred embodiment, the carrier vector has a baculovirus backbone. An internal
ribosome entry site (IRES) sequence may be placed between E2A and E4orf6 if
only a single promoter is to be used for these two proteins. Alternatively, each
helper function gene may be supplied with its own promoter. These genes may be
under the regulatory control of a variety of promoters, constitutive or inducible,
such as the CMV immediate-early promoter/enhancer or the MMTV LTR,
respectively. Whether the helper functions are provided on the carrier vector itself
or are provided by the host cells, the promoters regulating those genes may be constitutive or inducible.
The expression of the helper functions may be regulated by any of
the expression regulatory sequences known in the art or as described above,
including cis or trans regulation. The expression regulatory sequences may provide
for constitutive expression, inducible expression, tissue-, cell type- or differentiation
state-specific expression, or expression from the helper function protein's native
promoter. In a preferred embodiment, the native promoter of the helper function
protein is used. In another preferred embodiment, an inducible promoter of a helper
function protein is used. In another preferred embodiment, a constitutive promoter
of a helper function protein is used. In a further preferred embodiment, the
constitutive promoter is the CMV promoter. In another preferred embodiment, one
or more constitutive promoters are used for certain helper function proteins, and one or more native promoters are used for other helper function proteins.
In one embodiment, each protein or polypeptide required for helper
function is encoded by a nucleic acid whose expression is regulated by its own
promoter and polyadenylation signal, as well as optional sequences such as
enhancers. In another embodiment, a nucleic acid is transcribed to a single
transcript that encodes more than one protein or polypeptide required for helper
function. In this case, an IRES may be placed between the coding sequences of
each of the individual proteins or polypeptide to permit subsequent translation of
the polycistronic mRNA. If only a single polycistronic transcript is produced, only a single promoter, optional enhancer, and polyadenylation signal are required for
regulation of the transcription of the nucleic acid encoding the helper function. One
may also encode the helper function by using both monocistronic mRNAs that
encode single proteins and polycistronic mRNAs encoding multiple proteins.
In a preferred embodiment of the instant invention, the carrier vector
comprises a embedded recombinant AAV genome and helper functions. In a
further preferred embodiment, the helper functions comprise adenovirus El a, Elb
and E2a, and more preferably include E4ORF6 and VAI. In an even more preferred
embodiment, the helper functions are encoded by a single polycistronic transcript, and the promoter for the helper functions is a constitutive promoter, preferably the CMV promoter.
Other recombinant viruses would require different helper functions
or none at all, but in all cases those helper functions may be provided on the carrier
vector that carries the embedded recombinant viral genome, on a separate carrier
virus, on a different type of vector capable of transducing a mammalian host cell, or
is endogenously expressed in the mammalian host cell itself.
Mammalian Host Cells
Any type of mammalian host cell which can be adapted to cell
culture may be used to produce the recombinant viral genome. In general, a
mammalian host cell used in this invention is one that may be infected by a
nonmammalian carrier virus. The mammalian host cell may be one that may be
infected by a nonmammalian carrier virus that does not express a ligand encoded by a ligand nucleic acid, may be one that may be infected by a nonmammalian carrier
virus that expresses a ligand encoded by a ligand nucleic acid, or may be a cell that
is infected by a carrier vector that either expresses or does not express a ligand
nucleic acid. Alternatively, the mammalian host cell may be one that is not usually
infected by a carrier virus, but which can be transduced with a cellular receptor such
that it may bind to a nonmammalian host cell. For instance, a mammalian host cell
may be transduced with a growth factor receptor such that it can be infected by a
carrier virus that expresses the particular growth factor as its ligand.
In addition to the ability to be infected by the carrier virus, another preferred characteristic of the mammalian host cell is that it is able to uncoat the
nonmammalian carrier virus. A third preferred characteristic of the mammalian host
cell is its ability to replicate the recombinant virus at high levels. In a preferred
embodiment, the mammalian host cell is one that takes up the nonmammalian
carrier virus at high levels, uncoats the carrier virus efficiently, and replicates the
recombinant virus at high levels.
Appropriate mammalian host cells include, without limitation, CHO,
BHK, MDCK and various murine cells, e.g., 10T1/2 and WEHI cells, African green
monkey cells such as COS 1, COS 7, BSC 1, BSC 40, and BMT 10, and human
cells such as VERO, WI38, MRC5, A549, and HT1080 cells. In a preferred
embodiment, appropriate mammalian cell include 293 cells (human embryonic
kidney cells which express adenoviral Ela and Elb proteins), B-50 cells (HeLa cells
which express AAV rep and cap, see PCT US98/19463), 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).
In addition to the mammalian host cells listed above, other mammalian host cells may be used. One may determine whether a cell line would
be suited for use as a mammalian host cell by infecting the cell line with a carrier
virus containing all the required components to produce a recombinant virus,
culturing the cells under conditions in which recombinant virus is produced, and then measuring the titer of infectious recombinant virus that is produced. One may
then compare the titer of infectious virus produced in the potential host cell with the
titers produced by other host cells to determine whether the cell line is good for recombinant virus production.
Although the receptor(s) for nonmammalian carrier virus such as
baculovirus on both insect and mammalian cell is/are unknown, it is thought that the
baculovirus may bind to the cell, at least in part, via heparan sulfate expressed on
the cell surface. Without wishing to be bound by any theory, cells which express
high levels of heparan sulfate on their cell surface may be more easily infected by
carrier viruses, especially baculovirus, than cells which express low levels of
heparan sulfate on their cell surface. Thus, one method of identifying whether a
particular cell line is a potential mammalian host cell is to measure the level of
heparan sulfate on the cell surface.
Method of Making and Producing Carrier Viruses
The present invention includes methods of constructing the novel
carrier vectors described above and producing large quantities of the carrier vector.
This method comprises the steps of:
1. Modifying a nonmammalian virus backbone DNA, or a
replication-proficient portion thereof, by inserting one or more nucleic acid inserts
comprising 1 ) a recombinant viral genome comprising a transgene operably linked
to expression regulatory sequences and flanked by flanking elements; 2) nucleic acid
sequences encoding helper functions operably linked to expression regulatory
sequences; 3) nucleic acid sequences encoding replication and/or encapsidation
functions for the recombinant virus; 4) a ligand DNA operably linked to expression
regulatory sequences that are active in nonmammalian cells; and 5) regulatory
control sequences that regulate sequences in the nonmammalian virus backbone, a
odified nonmammalian virus backbone or a replication-proficient portion of the
backbone or modified backbone;
2 transducing the resulting carrier vector into nonmammalian host
cells, 3 growing the nonmammalian host cells under conditions in which
carrier virus is produced; and
4 collecting the carrier virus from the nonmammalian host cells
In a preferred embodiment, the carrier vector will be modified such
that it comprises the recombinant viral genome. In a more preferred embodiment,
the carrier vector will be modified such that it comprises the recombinant viral
genome and either or both of the nucleic acid inserts encoding the replication and/or
encapsidation functions and the helper functions required for production of a
recombinant virus In an even more preferred embodiment, the carrier vector will
be modified such that it comprises the recombinant viral genome, all of the nucleic
acid inserts encoding the replication and/or encapsidation functions and the helper
functions required for production of a recombinant virus, and the nucleic acid insert
encoding the ligand
The nonmammalian host cell may be any host cell known in the art
or described in Table 1 of U.S. Pat. No. 5,731,182. The nonmammalian virus
backbone DNA may be derived from any virus that infects nonmammalian species,
including those known in the art or described in Table 1 of U.S. Pat. No. 5,731,182
In a preferred embodiment, the nonmammalian backbone of the carrier vector is
derived from a baculovirus and the nonmammalian host cells are insect cells. In a
more preferred embodiment, the carrier virus produced by the baculoviral carrier
virus in the insect cells is produced at a high titer Preferably, when baculovirus is used, the titers of the carrier baculovirus produced in any embodiment are greater
than 108 pfu/ml in insect cells or IO9 pfu/ml, more preferably, the titers are greater
than 1010 pfu/ml or 1011 pfu/ml, and even more preferably, the titers are greater than
10'2 pfu/ml The instant invention also encompasses lysates and supernatants of
nonmammalian host cells comprising baculoviral carrier viruses having similar titers
The nonmammalian host cells comprising the carrier vector may be
grown by any method known in the art or as described herein Methods for
producing large amounts of nonmammalian viruses are well known in the art and are described in U S Pat No 5,871,986 The nonmammalian earner virus may be
purified from the supernatant produced by the nonmammalian host cells or from
lysed cells by any method known in the art or as described herein Methods for
collecting and purifying nonmammahan viruses are well known in the art and are
described in U S Pat No 5,871,986 A method of collecting and purifying the
nonmammalian viruses is described in Example 6
The carrier virus produced when the carrier vector is encapsidated
has the normal wild-type capsid optionally modified by addition of the ligand In
general, the expression of the ligand nucleic acid is regulated by expression
regulatory sequences which promote transcription and translation in the
nonmammahan host cells Such expression regulatory sequences may include a nonmammalian promoter active in the nonmammahan host cells of interest, and may
optionally include enhancer sequences, polyadenylation signals, or any other
expression regulatory sequences known in the art or described above In a
preferred embodiment, the other nucleic acid inserts in the nonmammahan backbone
may not be expressed or may be expressed at lower levels because their promoters
are inactive or less active in nonmammahan cells Because potentially toxic viral components, such as helper functions or rephcation encapsidation functions, are
either not expressed or expressed at lower levels, a high titer of carrier virus may be
produced in nonmammahan cells
Methods of Producing Recombinant Virus from the Carrier Vector
Another aspect of the instant invention is a method of producing
recombinant virus by using a carrier virus, produced by the method described
above, to infect mammalian cells and subsequently collecting and purifying the
recombinant virus from the mammalian cells The method comprises the steps of
1 Infecting mammalian host cells with a carrier virus, wherein he carrier virus optionally expresses a ligand on the surface of the carrier virus,
2 growing the infected mammalian host cells under conditions in
which the embedded recombinant viral genome is replicated, excised and
encapsidated, and
3 collecting the recombinant virus from the mammalian host cells
The mammalian host cells may be any mammalian host cell known in the art, described in the specification above under "Mammalian Host Cells," or
identified by the method descnbed under "Mammalian Host Cells" as an appropriate
host cell The mammalian host cell, prior to infection, may be one that expresses
one or more of the following: 1) replication and/or encapsidation functions (e.g., B-
50 cells or one of the retroviral cell lines described previously); 2) some or all necessary helper functions (e.g., 293 cells); and/or 3) an embedded recombinant
viral genome stably integrated in the mammalian host cell genome. Alternatively,
the mammalian host cell comprises none of these other elements before infection by
the carrier virus.
The mammalian host cells may be infected and grown by any method
known in the art or as described herein. Methods for infecting mammalian host
cells with nonmammalian viruses are described herein and in Barsoum et al., supra.
Once the mammalian host cell has been infected, the expression regulatory
sequences that are operably linked to any required replication and/or encapsidation
functions and helper functions are activated. Expression of the replication and/or encapsidation functions and helper functions, along with the mammalian host cell's
native transcriptional and translational components, permits replication, excision
and encapsidation of the embedded recombinant viral genome, thereby causing the
manufacture of the recombinant virus.
In general, the nonmammalian carrier virus is capable of infecting a mammalian cell, but the carrier virus will not replicate in the mammalian cell
because the components required for replication of the nonmammalian carrier virus
are not present in the mammalian cell. If the carrier virus comprises a ligand nucleic
acid, the expression regulatory sequences controlling ligand expression generally will not function in mammalian cells, such that ligand expression does not occur in
the mammalian host cell. However, if replication of the carrier virus or if
expression of the ligand is desired in the mammalian host cell, then additional expression regulatory sequences may be operably linked to the sequences required for replication, excision and/or packaging of the nonmammalian carrier virus and/or
expression of the ligand.
The recombinant virus may be purified from the supernatant
produced by the mammalian host cells or from lysed cells by any method known in
the art or as described herein. Methods for collecting and purifying various types of
recombinant viruses from mammalian host cells are well known in the art and are
described in PCT US97/15716. A method of collecting and purifying the
recombinant viruses is also described in Example 6.
As discussed above, in a preferred embodiment, one or more carrier
viruses comprises all those nucleic acid inserts required for production of recombinant virus in a particular mammalian host cell. Thus, if the host cell
comprises replication and encapsidation functions, then the carrier viruses comprise
the embedded recombinant viral genome and any necessary helper functions.
Similarly, if the host cell comprises the embedded recombinant viral genome, then one or more carrier viruses will comprise the required replication and encapsidation
functions and any necessary helper functions, while if the host cell comprises the
necessary helper functions, then one or more carrier viruses will comprise the
required replication and encapsidation functions and the embedded recombinant viral genome.
In a preferred embodiment, a single carrier virus comprises all of the nucleic acid inserts required for production of a recombinant virus in a particular
mammalian host cell. For example, if a recombinant AAV is to be produced in B-
50 cells, the carrier virus will comprise the embedded recombinant viral genome and
those helper functions required for AAV production. Similarly, if a recombinant
AAV is to be produced in a mammalian host cell that does not express any of the
required functions for AAV production, the carrier virus will comprise the
embedded recombinant viral genome, the replication and encapsidation functions
and the helper functions required for AAV production.
The method of producing recombinant virus described is useful
because it produces an essentially homogeneous recombinant virus that is free from helper virus and wild-type virus without purification. The recombinant virus is free
from helper virus because there are insufficient helper virus genes to produce a
mature helper virus. The recombinant virus is free of wild-type virus because
homologous recombination is avoided by a variety of techniques. For instance,
wild-type AAV produced through homologous recombination may be avoided using
several strategies. The rep/cap sequences and the embedded recombinant viral
genome may be positioned at separate loci on the carrier vector, minimizing the
likelihood of a recombination event. The rep/cap sequences and the embedded
recombinant viral genome also may be designed so that they have no regions of
homology. Additionally, because AAV is intolerant of packaging greater than 5.0
kb, one may incorporate a "stuffer" nucleic acid sequence to be inserted between a required sequence, such as rep or cap and its promoter. Thus, even if
recombination took place, the resulting AAV genome would be too large to
package and no wtAAV would be produced. In order to maintain the integrity of
translation of rep, the stuffer sequence may be constructed using splice donor and
acceptor sites, such that the resulting mRNA and ultimately the rep protein
produced would be unaffected. Similar methods may be employed for other types
of recombinant viruses to avoid recombination and production of wild-type virus. The method is also easily scaled to industrial production because it
may require only a single infection of mammalian host cells by a carrier virus that
can be produced in large amounts at high titers. In a preferred embodiment, the
recombinant virus produced by the instantly described method is produced at a high
titer. For recombinant AAV, the titer is preferably greater than IO4 particles per producing cell, more preferably, greater than 105 to IO6 particles per producing cell,
and even more preferably, greater than IO7 particles per producing cell. For
recombinant adenovirus, the titer is preferably greater than IO4 particles per producing cell, more preferably, greater than 105 particles per producing cell, and
even more preferably, greater than IO6 particles per producing cell. For recombinant herpesvirus, the titer preferably is greater than 1010 pfu per ml, more
preferably, greater than 1011 pfu per ml and even more preferably, greater than IO13
pfu per ml. For retroviruses, the titer preferably is greater than IO6 to IO7 colony
forming units (cfu) per ml, more preferably, 108 cfu per ml, and even more
preferably, 109 cfu per ml. The instant invention also encompasses lysates and
supernatants of mammalian host cells comprising recombinant viruses. These
lysates and supernatants differ from those produced by prior art methods because
they do not contain wild-type virus or helper virus.
In a preferred embodiment, the method is used to manufacture
recombinant AAV at high titers and in the absence of helper virus or wild-type
AAV The desired transgene, with appropriate expression regulatory sequences
operably linked thereto, is placed between the AAV ITRs, by means known in the
art In order to maintain the length of the insert at a length compatible with
eventual packaging, spacer DNA may optionally be inserted therein This
recombinant viral genome is then embedded in a baculovirus, in a non-essential
locus by means known in the art The required helper functions, replication and
encapsidation functions, and/or embedded recombinant viral genome may be placed
in the polyhedrin gene site, the plO gene site, or one could be placed at the
polyhedrin gene site and the other may be placed at the plO gene site (see Figure 1)
The baculovirus backbone also may be modified to comprise a ligand nucleic acid,
such as the VSV-G gene The baculovirus may also be modified to comprise rep and cap sequences and helper functions from adenovirus, comprising Ela, Elb,
E2a, E4ORF6 and VAI, or HSV genes UL5, UL8, UL52 and UL29 The carrier
vector is transduced into insect cells, such as Sf9 cells, the cells are grown under
conditions in which baculovirus is produced, and the baculovirus is collected and
purified The baculovirus is then used to infect mammalian cells, the mammahan
cells are grown under conditions in which the recombinant virus is replicated,
excised and encapsidated, and the recombinant AAV is collected and, optionally,
purified
In another preferred embodiment, the method is used to manufacture recombinant "gutless" adenovirus deleted of all adenoviral genes at high titers and
in the absence of helper virus or wild-type adenovirus Previously, one would make
a "gutted" adenovirus plasmid from the adenovirus genome in which all of the
adenovirus genes were removed except for the ITRs and the cis-acting packaging
signal Foreign DNA containing the transgene of interest, transcriptional regulatory
sequences, and, optionally, stuffer DNA would be added to obtain an insert of
approximately 36 kb The plasmid is designed such that a DNA cassette contains
the packaging signal upstream of the transgene and stuffer DNA, and the Ad ITRs
flank such DNA cassette. The plasmid was transfected into cells, such as 293 cells
A helper adenovirus lacking the adenoviral El and E3 genes, as well as sequences
within the adenoviral packaging signal was used to infect the 293 cells transfected
with the gutted Ad plasmid to provide replication and encapsidation functions in
trans Using this method, low levels of homologous recombination would rescue
the deletion in the helper Ad's packaging signal, thus both helper and "gutless"
adenovirus would be produced. CsCl gradients would have to be performed to
separate the helper adenovirus from the recombinant "gutless" adenovirus vector. In addition, other disadvantages included high levels of contaminating helper virus
and low yields of the gutless Ad vector.
Using the method of the instant invention, homologous
recombination resulting in generation of contaminating helper Ad can be avoided
Rather than using helper adenovirus, one may construct a carrier vector comprising
the adenoviral functions necessary for replication and packaging of the gutted Ad
genome. In one embodiment, the carrier vector contains the complete genome of
adenovirus without the ITR's, El, the packaging signal, and, optionally, without
E3 Then, one may infect mammalian cells with the carrier virus and transfect the
cells with the plasmid described above containing the transgene, ITRs and packaging signal In another embodiment, one may construct two separate carrier
vectors, one comprising helper adenoviral functions described above and the other comprising the gutted Ad construct containing the Ad ITR's, packaging signal and
the transgene cassette Alternatively, one may construct a single carrier vector comprising both the helper adenoviral functions and the transgene cassette
comprising the ITRs, packaging signal and transgene/stuffer DNA The adenoviral
functions and transgene cassette may be placed in the polyhedrin gene locus, the
p 10 gene locus, or one could be placed at the polyhedrin gene locus and the other may be placed at the pi 0 gene locus (see Figure 1).
In another preferred embodiment, the method is used to manufacture
recombinant herpesvirus amplicon vectors As discussed above, herpesvirus
amplicons require the "α" sequence for packaging, and the HSV origin of
replication Either the oris or oriL origin of replication may be used, but the oris
origin is preferred One may construct a carrier vector comprising a herpesvirus
amplicon In one embodiment, the cassette would contain, in the 5' to 3' direction,
the "α" sequence, followed by the transgene of interest, followed by the HSV origin
of replication, followed by an optional spacer, and followed by another "a"
sequence This may be inserted into either the polyhedrin or the plO gene loci in
baculovirus, for instance. The carrier virus that is subsequently produced may be
used to infect mammalian cells that have been coinfected with helper herpesvirus.
Alternatively, the helper herpesvirus functions may be placed on the same or a
separate carrier vector and used to infect the mammalian cells. Recombinant
herpesvirus amplicon vectors may then be isolated and purified from the mammalian
cells
Recombinant Virus Compositions
Another embodiment of the present invention is the recombinant
virus produced by the methods of the invention Unlike other preparations of
recombinant virus, the preparations produced by the methods of this invention yield
high titers of essentially homogeneous recombinant virus which is helper-free and
wild-type virus free The recombinant virus may be formulated as a
pharmacological composition for use for any form of transient and stable gene
transfer in vivo and in vitro The recombinant virus may be used for in vivo and ex
vivo gene therapy, genetic immunization, in vitro protein production and diagnostic
assays
For gene therapy, the recombinant virus may be introduced into cells
ex vivo or in vivo Where the virus is introduced into a cell ex vivo, the
recombinant virus may be used to infect a cell in vitro, and then the cell may
subsequently be introduced into a mammal (e g., into the portal vein or into the
spleen), if desired Alternatively, the recombinant virus may be administered to a
mammal directly, e g , intravenously or intraperitoneally. A slow-release device,
such as an implantable pump, may be used to facilitate delivery of the virus to a cell
Where the virus is administered to a mammal, the specific cells to be infected may
be targeted by controlling the method of delivery For example, intravascular
administration of the recombinant virus to the portal vein or to the hepatic artery
may be used to facilitate targeting the recombinant virus to a liver cell.
The recombinant virus produced by the above-described method
may be administered to a patient, preferably suspended in a biologically compatible
solution or pharmaceutically acceptable delivery vehicle. A suitable vehicle includes
sterile saline. Other aqueous and non-aqueous sterile suspensions known to be pharmaceutically acceptable carrier and well known to those of skill in the art may
be employed for this purpose.
The recombinant virus is administered in sufficient amounts to infect
the desired cells and provide sufficient levels of transduction and expression of the
selected transgene (or viral gene products in the case of a vaccine) to provide a
corrective effect without undue adverse or with medically acceptable physiological
effects, which can be determined by those skilled in the medical arts. Conventional
and pharmaceutically acceptable routes of administration include direct
administration to the target organ, tissue or site; intranasal; intravenous;
intramuscular; subcutaneous; intradermal; oral and other parenteral routes of
administration. Routes of administration may be combined, if desired.
Dosages of the recombinant virus will depend primarily on factors
such as the type of recombinant virus (i.e., whether the virus is AAV, adenovirus,
retrovirus, etc.), the condition being treated and the selected gene. The dosage may
also vary depending upon the age, weight and health of the patient. For example,
an effective human dosage of a recombinant adenovirus is generally in the range of from about 0.5 ml to 50 ml of saline solution containing adenovirus at
concentrations of 1 x 107 or 1 x 108 or 1 x IO9 or 1 x 1010 or 1 x IO11 or 1 x IO12 or
1 x I O13 or 1 x 10!4 or 1 x IO15 particles per dose administered. 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.
The following examples of the present inventions are illustrative
only, and are not intended to limit the scope of the invention.
EXAMPLE 1 Cell Line Maintenance and Virus Propagation
The human embryonic kidney cell line 293 (ATCC CRL 1573) was
maintained in Dulbecco's Modification of Eagle's Medium (DMEM; GIBCO BRL)
supplemented with 10% FBS (Hyclone) and 50 μg of penicillin, 50 μg of
streptomycin, and 10 μg of neomycin/ml (GIBCO BRL). Insect cell line IPLB-Sf21
(CLONTECH Laboratories, Inc.) was maintained in SF900-II medium (GIBCO
BRL) supplemented with 10% FBS and 50 μg of penicillin, 50 μg of streptomycin,
and 10 μg of neomycin/ml. Human adenovirus type 5 (ATCC VR-5) was
propagated on 293 cells and purified through CsCl gradient centrifugation (Jones
and Shenk, 1978).
EXAMPLE 2 Recombinant Plasmid Construction
Standard DNA recombinant techniques were employed to create
recombinant plasmids (Sambrook et al, 1989). The Rep and Cap sequence of pAV2
(ATCC 37216) between the Drain site upstream of the p5 promoter and the Ncol
site downstream of the polyadenylation signal was removed The Rep and Cap
sequence was replaced through multiple cloning steps with a cassette containing
GFP under the control of elongation factor 1 alpha (EFlα) promoter to create
pAV2cisEFGFP (Fig 2) The entire cassette containing both AAV ITRs and the
GFP gene was then cloned into the Spel and Bglll sites of BV-CZPG (baculovirus
shuttle plasmid with VSV-G gene under control of polyhedrin promoter, kindly
provided by Dr Jim Barsoum of Biogen, Ine ) through multiple cloning steps to
obtain pBV-cisEFGFP (Fig 3)
Adenovirus helper genes E2A, E4ORF6, and VAI were subcloned
from Ad5 DNA Briefly, E4ORF6 was first inserted into the Smal and Xbal sites of
pIRESlneo (CLONTECH Laboratories, Ine ) to obtain pIRESORFό Next, a
Sau3AI-BsrGI fragment containing E2A coding sequences was inserted into the BamHI/BstXI sites of the plasmid pIRESORF6 through multiple cloning steps to
obtain the plasmid pE2 A ORF6 In this construct, E2A and E4ORF6 genes are
separated by an encephalomyocarditis virus (ECMV)-derived IRES, and both genes
are under the transcriptional control of a single human cytomegalovirus (CMV)
promoter upstream of the E2A gene Next, a NcoI-BamHJ fragment of Ad5 DNA
containing the VAI gene was inserted into the Xhol site of pE2AiORF6 through
blunt-end cloning to obtain pE2AiORF6-VAI The entire cassette containing
CMV-E2AORF6-VAI was cloned into the Hpal and Spel sites of the baculovirus shuttle plasmid BV-CZPG to obtain pBV-EiOV (Fig. 2).
AAV-2 rep and cap genes located between a Dra III site, which is
upstream of the AAV-2 p5 promoter and a Bsal site, which is downstream of the
polyadenylation signal (Fig 2), were cloned into the Spel and Pad sites of pBV-
EiOV through multiple cloning steps to obtain pBV-EiOV-RC (Fig 3) Ad5 El
and cisEFGFP were cloned into pBV-EiOV through multiple steps to obtain the
pBV-EiOV-cisEFGFP-El plasmid (Fig 3) Plasmid pAc-cisEFGFP was
constructed by inserting a cassette containing the GFP gene flanked by AAV-2
ITRs into pAcUWl (Pharmingen) through several cloning steps The Xbal-Sspl
fragment of EBVOπ from pEBVHisA (Invitrogen) was inserted into pBV-
cisEFGFP and pBV-EiOV-RC to create pBV-cisEFGFP-EBVOn (Fig 3) and
pBV-EiOV-RC-EBVOri (Fig 3) AAV-2 rep and cap genes were cloned into
pAdΔF6 (plasmid carrying Ad helper genes E2A, the entire E4, and VAI, kindly
provided by Dr Guangping Gao of the University of Pennsylvania) to obtain
pAdΔF6-RC (Fig 3)
Reference to a construct preceded by a "p" refers to a plasmid, while
reference to a construct without a "p" preceding it refers to a virus For example,
pBV-EiOV-RC refers to a plasmid, while BV-EiOV-RC refers to a modified
baculovirus
EXAMPLE 3 Transfection of 293 Cells and Selection for 293-CG3 Stable Cell Line
293 cells were grown to -70% confluency in 6-cm dia tissue culture
dishes and co-transfected overnight with 1 μg pIRESlneo and 10 μg
pAV2cιsEFGFP by the calcium phosphate transfection method (Sambrook et al ,
1989) Cells were fed with fresh medium containing 10% FBS and cultured for 24 hours Following trypsinization, cells were seeded at a 1 20 dilution in fresh
medium containing 10% FBS After incubation for another 24 hours, fresh medium
containing 1,250 μg/ml of G418 (GIBCO BRL) was added to the cell monolayer to
select for G418-resιstant cells The medium containing G418 was replaced every 3-
4 days until most of the original G418-resιstant cell colonies had formed A total of
fifty colonies were picked, six of which demonstrated constitutive GFP expression
These six clones were expanded in the presence of G418 and tested for their ability
to rescue functional rAAV by transfection with plasmid pBV-EiOV-RC Normally, when a clone was established, it was maintained in G418-contamιng medium for 3
to 5 passages to ensure that all nonresistant cells had been killed Then, the cells
were maintained in G418-free medium One cell clone, 293-CG3, showed high
efficiency of rAAV rescue and was expanded and used for further experiments
EXAMPLE 4 Functional Test of 293-CG3 for rAAV Production
Several plasmids were used to test the efficiency of 293-CG3 cells
for rAAV production The cells were first seeded on 6-well plates at a density of 1
X 106 cells/well at 2 to 4 hours before transfection The cells in each well received
5 μg of plasmid DNA in a final volume of 167 μl of CaPO4 (Sambrook et al , 1989)
After incubation for 16 hours, cells were fed with fresh medium Three days later,
cells were harvested and rAAV titers (transducing units) were determined The
results are presented in Table 1 as an average of values determined from two
separate experiments. They indicate that the 293-CG3 cell line is very efficient for rAAV production, producing approximately 30 to 170 transducing units of rAAV
per cell depending on the configuration of helper plasmid used.
Table 1.
EXAMPLE 5 Generation of Recombinant Baculoviruses
To create VSV-G pseudotyped recombinant baculoviruses, the
BacPAK baculovirus expression system (CLONTECH Laboratories, Inc.) was
used. Plasmid DNA, one of pBV-EiOV-RC, pBV-cisEFGFP, or pBV-EiOV-
cisEFGFP-El, was cotransfected with Bsu36I-digested BacPAKό DNA into Sf21
cells according to the manufacturer's protocol. The medium was harvested 3 days
after transfection and recombinant baculoviruses were screened on 96-well plates by
limited dilution assay. Briefly, the medium harvested from transfection was diluted
to I O"2, IO"3, IO"4, and 10"5 each in 10 ml of insect medium containing Sf21 cells at 2
X 105 cells/ml. The mixture was then plated on 96-well plates at 100 μl/well.
After infection for 5-7 days, cells were examined for signs of viral
infection (cell fusion mediated by VSV-G expression; see Eidelman et al., 1984).
The weels that showed viral infection in the lowest dilution were marked and the
virus harvested (Chen et al., 1994). The cells were lysed and used for DNA
hybridization to verify the presence of recombinant DNA Positive clones were
amplified into 10 ml, and 1 ml of each clone was used to transduce 293 cells grown in 6-well plates The 293 cells were transfected with a plasmid carrying the
elements not provided by the recombinant baculovirus for rAAV rescue After
transduction for 3 days, cells were lysed and the lysates were used to transduce 84-
31 cells (E1/E4 double complementing cell lines derived from 293 cells, see Fischer
et al , J Virol , 70 8934-8943, 1996) The expression of a marker gene indicated
the rescuing of rAAV The cloned that could best support rAAV rescue was
screened for 3 to 4 more rounds in order to obtain pure recombinant baculovirus
and tested for their support of rAAV rescue Functional clones were further
screened on 96-well plates for 3 to 4 rounds to obtain pure recombinant
baculoviruses
To create non-VSV-G pseudotyped recombinant baculoviruses, a Baculovirus Expression Vector System (Pharmingen) was used Plasmid DNA was
co-transfected with baculoviral DNA into Sf21 cells according to manufacturer's
protocol Recombinant baculovirus was screened on 96-well plates the same as
described for the VSV-G pseudotyped baculoviruses above except that X-gal staining was used to distinguish recombinant baculovirus from wild type
baculovirus
EXAMPLE 6 Production of rAAV by Using the Methods of this Invention
Method to Transduce Cells
For transduction, recombinant baculoviruses were first pelleted by
centrifugation at 4"C at 20,000 rpm for 30 minutes. The pellets were then
resuspended in serum-free DMEM. The medium was removed from the cell
monolayer and baculovirus was added to the cells. After incubating for 8-16 hours,
cells were fed with fresh medium containing 10% FBS. Cells were harvested 72
hours after transduction. Baculovirus-transduced cells were harvested and lysed in
DOC lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM MgCl2, 0.5% sodium
deoxycholate) by sonication on ice water (three sonication pulses for 1 minute
each). Cell debris was removed by centrifugation at 13,000 rpm for 5 minutes at
4°C and the supernatant was collected. The supernatant was used for rAAV
titration as described in Example 7.
Transduction of 293 cells by Baculoviruses BV-EiOV-RC and BV-cisEFGFP
BV-EiOV-RC provides Ad helper genes E2A, E4ORF6, and VAI as well as AAV rep and cap genes. BV-cisEFGFP provides the AAV vector sequence
with both AAV ITRs flanking the marker gene GFP. 293 cells express Ela and
Elb. Thus, transduction of 293 cells with both BV-EiOV-RC and BV-cisEFGFP
provides to the cells the embedded AAV viral genome comprising the GFP transgene operably linked to the EFlα promoter and flanked by the AAV ITRs; a
VSV-G ligand; helper functions comprising Ela, Elb, E2a, E4ORF6, and VAI; and
the replication and encapsidation functions, rep and cap. These functions allow the
cells to produce recombinant AAV.
Transduction of 29 -CG 3 Cells by Baculovirus BV-EiOV-RC
Because the AAV vector was stably integrated in the 293-CG3 cells,
only BV-EiOV-RC was needed to provide a ligand nucleic acid, helper functions
and replication and encapsidation functions to produce recombinant AAV
Transduction of Rep-Cap Expressing Cells by Baculovirus BV-EiOV-cisEFGFP-El
The baculovirus BV-EiOV-cisEFGFP-El provides the Ad helper
genes El, E2A, E4ORF6, and VAI, as well as the AAV vector with both AAV-
ITRS flanking the marker gene GFP. The AAV rep-cap genes are provided by
stable rep-cap cell lines such as B50 (Gao et al. 1998).
EXAMPLE 7 Titration of rAAV Produced by Baculovirus Transduction
An rAAV lysate from baculovirus-transduced cells prepared as
described in Example 6, was diluted at IO"2, IO"3 and IO"4 with DMEM containing
10% FBS and used for the titration assay. 24-well plates were first coated with
0 1% gelatin for 30 minutes and then plated with 2 X 105 cells/well of 293 -based
84-31 cells (Fischer, et al., 1996). After 3 to 4 hours of incubation, the cells were
infected with adenovirus at 100 particles/cell for 30 minutes (adenovirus helps the
conversion of single stranded AAV into double stranded AAV and is widely used
for AAV titration), and then the diluted rAAV lysate was added After transduction
by the rAAV for 24 hours, the cells were fixed with 4% paraformaldehyde in PBS
for 30 minutes The paraformaldehyde was replaced with PBS, and GFP-
expressing cells were counted under by fluorescent microscopy
EXAMPLE 8
Production of rAAV Using VSV-G Pseudotyped Baculovirus
Recombinant baculovirus BV-EiOV-RC was used to transduce 293-
CG3 cells for rAAV production The cells were first plated on 6-well plates at a
density of 1 X IO6 cells/well at 2 to 4 hours prior to transduction Baculovirus BV-
EiOV-RC was concentrated by centrifugation at 20,000 rpm at 4°C for 30 minutes,
resuspended in serum-containing DMEM, and then added to the cells at the
indicated amounts as shown in Table 2 After incubation for 16 hours, cells were
fed with fresh medium Following transduction for a total of 3 days, cells were
harvested and rAAV titers (transducing units) determined The results are
presented in Table 2 as an average of values determined from two separate
experiments They results indicate that recombinant baculovirus carrying Ad helper
and AAV rep-cap genes can successfully transduce 293-CG3 cells and produce rAAV By increasing the multiplicity of infection (moi) of input baculovirus, higher titers of rAAV were produced
Table 2
EXAMPLE 9
Transduction Efficiency of Different Mammalian Cell Lines by VSV-G Pseudotyped and Non-Pseudotvped Baculoviruses
In order to identify a suitable cell line that is efficiently transduced by
baculovirus, a number of cell lines were tested. Cells were seeded on 6-well plates
and grown to -80% confluency. Baculoviruses were added to the cells at the
indicated moi's in serum-free DMEM, incubated with the cells overnight, and
replaced with fresh medium 12-15 hours later. GFP-expressing cells were scored as
a percentage of all cells in the monolayer at 48 hours post-transduction by the
recombinant baculovirus.
The results presented in Table 3 indicate that, in general, VSV-G pseudotyped baculovirus (BV-cisEFGFP) transduce mammalian cells much more
efficiently than the non-pseudotyped baculovirus (Ac-cisEFGFP). However, HepG2
and Saos-2 cell lines were found to be more transducible than HeLa and 293 cell
lines by baculovirus. Insertion of Ad El genes into the chromosome of these cell,
similar to the scenario in 293 cells, should further facilitate production of rAAV
from these cells using the recombinant baculoviruses cited in previous examples. It
is noteworthy that Saos-2 cells are highly permissive for infection by baculovirus
irrespective of the presence or absence of the VSV-G glycoprotein on the baculoviral coat. The use of this cell line could provide an advantage for rAAV
production using non-pseudotyped baculovirus
All documents cited above are incorporated by reference herein.
Numerous modifications and variations of the present invention are included in
the above-identified specification and are expected to be obvious to one of skill
in the art. Such modifications and alterations to the processes of the present
invention are believed to be encompassed in the scope of the claims appended
hereto.
Table 3
References
1 Jones N, Shenk T. Isolation of deletion and substitution mutants of
adenovirus type 5 Cell 1978 Jan;13(l): 181-8
2 Sambrook, J , Fritsch, E.F., and Maniatis, T "Molecular Cloning — A
Laboratory Manual" Cold Spring Harbor Laboratory Press. 1989
3 Gao GP, Qu G, Faust LZ, Engdahl RK, Xiao W, Hughes JV, Zoltick PW,
Wilson JM High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus Hum Gene Ther 1998 Nov l;9(16).2353-62
4 Fisher KJ, Gao GP, Weitzman MD, DeMatteo R, Burda JF, Wilson JM
Transduction with recombinant adeno-associated virus for gene therapy is limited
by leading-strand synthesis. J Virol 1996 Jan;70(l):520-32.
5 Eidelman, O , Schlegel, R., Tralka, T.S., and Blumenthal, R. J Biol Chem 1996, 259 4622-4628.
6 Chen, H. and Padmanabhan, R. Biotechniques 1994, 17.40-42
Claims
1 A carrier vector for the manufacture of a recombinant virus,
wherein the carrier vector comprises a nonmammalian virus backbone, or a portion
or a modification thereof which is capable of replication in a nonmammalian cell
2 The carrier vector of claim 1 further comprising one or more of
the following elements' 1) an embedded recombinant viral genome, 2) nucleic acid
sequences which encode proteins required for replication and encapsidation of the
recombinant viral genome, 3) nucleic acid sequences encoding helper functions, 4)
nucleic acid sequences that encode a ligand that can interact with a mammalian cell,
and 5) regulatory control sequences that regulate sequences in the nonmammalian
virus backbone or in a replication-proficient portion or modification thereof
3 The carrier vector according to claim 2 which comprises nucleic
acid sequences that encode a ligand that can bind to a mammalian cell receptor
4 The carrier vector according to either of claims 1, 2 or 3 which
further comprises an embedded recombinant viral genome
5. The carrier vector according to either of claims 1, 2 or 3 which
further comprises nucleic acid sequences which encode proteins required for
replication and encapsidation of the recombinant viral genome.
6. The carrier vector according to either of claims 1, 2 or 3 which
further comprises nucleic acid sequences encoding helper functions.
7. The carrier vector according to either of claims 1, 2 or 3 which
further comprises regulatory control sequences that regulate expression of nucleic acid sequences in the nonmammalian virus backbone or in a replication-proficient
portion or modification thereof.
8. The carrier vector according to either of claims 1, 2 or 3 which
further comprises nucleic acid sequences which encode proteins required for
replication and encapsidation of the recombinant viral genome and nucleic acid
sequences encoding helper functions.
9. The carrier vector according to either of claims 1, 2 or 3 which
further comprises an embedded recombinant viral genome and nucleic acid sequences which encode proteins required for replication and encapsidation of the
recombinant viral genome and/or nucleic acid sequences encoding helper functions.
10. The carrier vector according to either of claims 1, 2 or 3 which
comprises all those elements which are required by a mammalian host cell to
produce an infectious recombinant virus.
11. The carrier vector according to claim 2 or 3, wherein the nucleic acid sequences that encode a ligand that can interact with a mammalian cell are
under the regulatory control of a non-mammalian promoter.
12 The carrier vector according to claim 11, wherein the ligand can
bind to a mammalian cell receptor.
13 The carrier vector according to claim 2 or 3, wherein the
embedded recombinant viral genome comprises flanking sequences derived from an
adeno-associated virus (AAV), an adenovirus, a retrovirus or a herpesvirus.
14. The carrier vector according to claim 13, wherein the embedded
recombinant viral genome comprises flanking sequences derived from AAV and the carrier vector further comprises helper sequences encoding a protein providing a
helper function required for replication of AAV.
15. The carrier vector according to claim 14, wherein said helper
sequences are derived from adenovirus (Ad) DNN herpes simplex virus (HSV)
type I or type II DNA, pseudorabies virus (PRV), cytomegalovirus (CMV) or vaccinia virus.
16. The carrier vector according to claim 15, wherein said helper
sequences encode at least one gene product selected from the group consisting of
adenoviral genes El A, E1B, E2A, E4orf6 and VAI, or at least one gene product selected from the group consisting of HSV type 1 genes UL5, UL8, UL52, and
UL29
17 The carrier vector according to claim 14, further comprising a
nucleic acid sequence encoding the AAV rep and cap proteins
18 The carrier vector according to claim 13, wherein the embedded
recombinant viral genome comprises flanking sequences and packaging signals
derived from adenovirus.
19 The carrier vector according to claim 13, wherein the embedded
recombinant viral genome comprises a herpesvirus "a" packaging sequence and a
herpesvirus origin of replication.
20 The carrier vector according to claim 2 or 3, wherein the
embedded recombinant viral genome comprises a transgene whose expression is regulated by expression regulatory sequences operably linked to said transgene.
21 A method for producing a carrier virus, comprising the steps of:
a) modifying a nonmammalian virus backbone DNA, or a
replication-proficient portion or modification thereof, by inserting one or more
nucleic acid inserts comprising
i) a recombinant viral genome comprising a transgene operably linked to expression regulatory sequences and flanked by flanking
elements,
ii) nucleic acid sequences encoding helper functions operably
linked to expression regulatory sequences, iii) nucleic acid sequences encoding replication and/or
encapsidation functions for the recombinant virus, iv) a ligand DNA operably linked to expression regulatory
sequences that are active in nonmammalian cells, and
v) regulatory control sequences that regulate sequences in
the nonmammalian virus backbone, a modified nonmammalian virus backbone or a
replication-proficient portion of the backbone or modified backbone;
b) transducing the resulting carrier vector into nonmammalian host
cells, c) growing the nonmammalian host cells under conditions in which
carrier virus is produced; and d) collecting the carrier virus from the nonmammalian host cells.
22 The method according to claim 21, wherein the nonmammalian
virus backbone is derived from baculovirus and the nonmammalian host cells are
insect cells.
23. A lysate or supernatant comprising the carrier virus produced by
the method according to either of claims 21 or 22. 24 The method according to either of claims 21 or 22, further
comprising the step of purifying the carrier virus
25 A purified preparation of carrier virus produced by the method according to any one of claims 21, 22 or 24
26 A method for producing a recombinant virus, comprising the
steps of
a) infecting mammalian host cells with a carrier virus, wherein the
carrier virus optionally expresses a ligand on its surface;
b) growing the infected mammalian host cells under conditions in
which the embedded recombinant viral genome is replicated, excised and
encapsidated, and
c) collecting the recombinant virus from the mammalian host cells
27 The method according to claim 26, wherein the mammalian host
cells are selected from CHO, BHK, MDCK, 10T1/2, WEHI cells, COS, BSC 1, BSC 40, BMT 10, VERO, WI38, MRC5, A549, HT1080, 293, B-50, 3T3,
NIH3T3, HepG2, Saos-2, Huh7 or HeLa cells.
28 A lysate or supernatant comprising the recombinant virus produced by the method according to either of claims 26 or 27. 29 The method according to either of claims 26 or 27 further comprising the step of purifying the recombinant virus from the mammalian host
cells
30 A purified recombinant virus produced by the method according
to any one of claims 26, 27 or 29.
31 A pharmaceutical composition comprising the carrier virus
according to claim 25 or the recombinant virus according to claim 30 and further
comprising a pharmaceutically acceptable carrier.
32 A method for transient or stable gene transfer of a desired
transgene to a mammalian cell, comprising the step of infecting the mammalian cell
with the recombinant virus according to claim 30.
33 The method according to claim 32, wherein said transient or stable gene transfer is for genetic immunization, correction of genetic defects or
production of proteins in vitro, in vivo, or ex vivo.
34 A method of using a recombinant virus comprising a point
mutation or deletion as a vaccine, comprising the steps of producing an attenuated
replication-proficient recombinant virus or a replication-deficient recombinant virus
by the method of claim 26 and administering the recombinant virus to a patient in a dose effective to induce an immunogenic response.
35 The pharmaceutical composition according to claim 31, wherin
the carrier virus further comprises adenoviral sequences required for replication and
encapsidation of the recombinant adenovirus.
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US9333249B2 (en) | 2006-02-09 | 2016-05-10 | Educational Foundation Jichi Medical University | Recombinant baculovirus vaccine |
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