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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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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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  • 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
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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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  • 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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  • 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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  • 2000-05-25 JP JP2001500792A patent/JP2003501042A/ja active Pending
  • 2000-05-25 WO PCT/US2000/014481 patent/WO2000073480A1/en active IP Right Grant
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