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EP0797454A1 - Vecteurs adenoviraux ameliores et cellules productrices - Google Patents

Vecteurs adenoviraux ameliores et cellules productrices

Info

Publication number
EP0797454A1
EP0797454A1 EP95943705A EP95943705A EP0797454A1 EP 0797454 A1 EP0797454 A1 EP 0797454A1 EP 95943705 A EP95943705 A EP 95943705A EP 95943705 A EP95943705 A EP 95943705A EP 0797454 A1 EP0797454 A1 EP 0797454A1
Authority
EP
European Patent Office
Prior art keywords
adenoviral
vector
dna sequence
particles
free
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.)
Withdrawn
Application number
EP95943705A
Other languages
German (de)
English (en)
Other versions
EP0797454A4 (fr
Inventor
Michael Kadan
Mario Goziglia
Bruce Trapnell
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genetic Therapy Inc
Original Assignee
Genetic Therapy Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genetic Therapy Inc filed Critical Genetic Therapy Inc
Publication of EP0797454A1 publication Critical patent/EP0797454A1/fr
Publication of EP0797454A4 publication Critical patent/EP0797454A4/fr
Withdrawn legal-status Critical Current

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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/108—Plasmid DNA episomal vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/70—Vectors containing special elements for cloning, e.g. topoisomerase, adaptor sites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses

Definitions

  • This invention relates to adenoviral vectors wherein the adenoviral genome has been modified to reduce the host immune and inflammatory responses to the vector. More particularly, this invention relates to adenoviral vectors which are free of all or a part of the adenoviral El and E4 DN ⁇ sequences, or are free of all or a part of the adenoviral El and E2 DN ⁇ sequences or are free of all or a portion of each of the El, E2, and E4 regions and further may or may not contain some of the E3 region genes operationally linked to a functional promoter, and to producer cells for generating adenoviral particles from such vectors.
  • Adenoviru ⁇ genomes are linear, double-stranded DNA molecules about 36 kilobase pairs long. Each extremity of the viral genome has a short sequence known as the inverted terminal repeat (or ITR) , which is necessary for viral replication.
  • ITR inverted terminal repeat
  • the well-characterized molecular genetics of adenovirus render it an advantageous vector for gene transfer.
  • the knowledge of the genetic organization of adenoviruses allows substitution of large fragments of viral DNA with foreign sequences.
  • recombinant adenoviruses are structurally stable and no rearranged viruses are observed after extensive amplification. / S95/15947
  • Adenoviruses thus may be employed as delivery vehicles for introducing desired genes into eukaryotic cells, whereby the adenovirus delivers such genes to eukaryotic cells by binding cellular receptors, internalizing via coated pits, disrupting endosomes, and releasing particles to the cytoplasm followed by nuclear translocation and molecular expression of the adenovirus genetic program.
  • an adenovirus genome includes the El region, the E2 region, the E3 region, the E4 region, and the major late region as well as several other minor regions.
  • the El region provides functions associated with transformation, transcription upregulation, and control of replication.
  • the E2 region is essential for DNA replication, and other viral functions including transcriptional regulation.
  • the E3 region provides for the adenovirus' defense against the host's immune system.
  • the E3 region encodes proteins which inhibit the host cell's ability to present viral proteins as foreign antigens.
  • the E4 region is essential for several important viral functions, including control of late gene expression, shutting down host cell function by the virus, and virus replication.
  • the major late region encodes the major adenoviral structural proteins.
  • Adenoviral vectors have been constructed in which at least a majority of the El and E3 sequences have been deleted.
  • the El deletion renders the vector replication defective
  • the E3 region deletion provides space for insertion of the desired gene into the adenoviral vector without exceeding the maximum acceptable size for a packageable genome.
  • Applicants have found that when such vectors have been placed into target cells or organs in vivo, however, a sharp decrease in expression of the desired gene results at about 1 to 3 weeks post-infection.
  • an inflammatory response may develop in a target organ, and that the adenovirus vector may induce a cytotoxic T-lymphocyte (CTL) response directed against the vector-transduced host cells. This CTL response has the effect of eliminating the host vector transduced cells from the host. Also, the host may develop a neutralizing antibody response to such adenoviral vectors.
  • CTL cytotoxic T-lymphocyte
  • Figure 1 is a schematic of the construction of plasmid pSE280-El;
  • Figure 2 is a map of plasmid pMAMneo-Luc,*
  • Figure 3 is a schematic of the construction of plasmid pMAMneo-El;
  • Figure 4 is a schematic of the construction of plasmid pGRE5-El from pSE280-El and pGRE5-2/EBV;
  • Figure 5 is a map of plasmid pGRE5-El
  • Figure 6 a schematic of the construction of plasmid pMAMneo-E2A
  • Figure 7 is a map of plasmid pSE380
  • Figure 8 is a schematic of the construction of plasmids pSE380-E4 and pSE380-ITR/E4;
  • Figure 9 is a schematic of the construction of plasmid pSE380GRE5/E4
  • Figure 10 is a map of plasmid pTZ18R
  • Figure 11 is a schematic of the construction of plasmids pTZE2A and pTZdlE2A;
  • Figure 12 is a schematic of the construction of plasmid pSE380-E3 + ;
  • Figure 13 is a schematic of the construction of plasmids pSE380-E3 + E4 + and pTZE3 + E4 + ;
  • Figure 14 is a schematic of the construction of plasmids pTZE3E4 + , pSE280E3 ⁇ 4 + , and pSPORT-1 E3 ⁇ 4 + ;
  • Figure 15 is a map of plasmid pSPORT-1
  • Figure 16 is a schematic of the construction of plasmid pSPORT-lE2AE3'E4 + ;
  • Figure 17 is a map of plasmid pBR322,*
  • Figure 18 is a schematic of the construction of plasmids pBR322-E2A ' E3'E4 + and pBRAd5-E2AE3 ⁇ 4 + ;
  • Figure 19 is a schematic of the construction of plasmids pSE380-E4- and pSE380E3 + E4 " ;
  • Figure 20 is a schematic of the construction of plasmid pSE380E3 + E4 (2) ;
  • Figure 21 is a schematic of the construction of plasmid pSE380E3 ⁇ 4 (2) ;
  • Figure 22 is a schematic of the construction of plasmid pSPORTl/E2A'E3 ⁇ 4 ' (2) ,*
  • Figure 23 is a schematic of the production of an adenoviral vector having the genotype El + , E2A “ , E3 " , E4 + ;
  • Figure 24 is a map of pAvS6
  • Figure 25 is a schematic of the construction of an adenoviral vector having the genotype El " , E2A ' , E3 ' , E4 + by homologous recombination,*
  • Figure 26 is a schematic of the production of an adenoviral vector having the genotype El “ , E2A “ , E3', E4 + by in vitro ligation and transfection,*
  • Figure 27 is a schematic of the production of an adenoviral vector having the genotype El “ , E2A “ , E3 “ , E4 + by homologous recombination,*
  • Figure 28 is a schematic of the vector Av3nLacZ
  • Figure 29 is a schematic of the production of an adenoviral vector having the genotype El + , E4 " ,*
  • Figure 30 is a schematic of the production of an adenoviral vector having the genotype El ' , E2 + , E3 + , E4 ' by homologous recombination
  • Figure 31 is a schematic of the production of an adenoviral vector having the genotype El " , E2 + , E3 + , E4 ' by in vitro ligation and transfection,*
  • Figure 32 is a schematic of the generation of Av4nLacZ by homologous recombination
  • Figure 33 is a schematic of the generation of Av4nLacZ by in vitro ligation,*
  • Figure 34 is a schematic of the generation of an Av4 (fourth generation adenovirus) type vector containing a transgene,*
  • Figure 35 is a schematic of the construction of plasmid pAvS6-CFTR
  • Figure 36 is an immunoprecipitation blot for adenoviral hexon expression in A549 cells infected with an Avl type vector (having a deletion of all or part of the El region) , an Av2 vector (having a deletion of all or part of the El region and a temperature-sensitive mutation in the E2a gene) , or an Av3 vector or Ad dl327,*
  • Figure 37 is an immunoprecipitation blot for adenoviral hexon expression in El + E2 + cells or El + cells infected with an Avl vector, an Av2 vector, or an Av3 vector;
  • Figure 38 is a blot showing adenovirus vector DNA replication in El + E2 + or El + cells infected with an Avl vector, an Av2 vector, or an Av3 vector.
  • the present invention provides for an improved adenoviral vector wherein the adenoviral genome has been modified to reduce the host immune and inflammatory response to the vector.
  • Such vector has a reduced viral DNA replication potential and reduced expression of adenoviral genes (e.g., hexon) in comparison to existing adenoviral vectors.
  • modification(s) to the adenoviral genome may be effected through deletion(s) and/or mutation(s) of portion(s) of the adenoviral genome.
  • the adenoviral vectors described herein include several general types or generations, each of which are described below.
  • These vectors include an adenovirus DNA genome containing an adenovirus 5'ITR; an adenovirus 3'ITR; an adenovirus encapsidation signal; at least one DNA sequence encoding a protein or polypeptide of interest; and a promoter controlling the at least one DNA sequence encoding a protein or polypeptide of interest.
  • adenoviral vectors (Av3 vectors) contain a deletion of all or part of the El region and also a deletion of all or a part of the E2 region (preferably all or part of the E2a region) .
  • a second generation of adenoviral vectors contain a deletion of all or part of the El region and a deletion of all or a part of the E2 region (preferably all or part of the E2a region) , and a deletion of all or a part of the E4 region.
  • a third generation of adenoviral vectors contain a deletion of all or a part of the El region, and a deletion of all or part of the E4 region. Each of these vectors may or may not contain some part of the E3 region operationally linked to a promoter which controls its expression.
  • the DNA sequence encoding a protein or polypeptide of interest, which is linked operationally to a promoter controlling its expression is sometimes hereinafter referred to as a transgene.
  • Cell lines containing adenovirus structural genes under the control of an inducible promoter are referred to as iEl (El genes) ,* iE2a (E2a genes) ,* iEl/E2 (El and E2 genes); (i)El/E4 (El and E4 genes); or iEl/E2a/E4 (El, E2, and E4 genes).
  • an adenoviral vector which includes an adenoviral 5' ITR; an adenoviral 3' ITR,* an adenoviral encapsidation signal; at least one DNA sequence encoding a protein or polypeptide of interest; and a promoter controlling the DNA sequence(s) encoding a protein(s) or polypeptide(s) of interest.
  • the adenoviral vector is free of all or a portion of the adenoviral El (preferably at least all or a portion of Ela) and E4 DNA sequences.
  • the adenoviral vector also is free of all or a portion of adenoviral Elb DNA sequence.
  • Such a vector is sometimes referred to as an adenoviral E1E4 " vector.
  • portion means that a portion of the DNA sequence of a region (e.g., El, E2, E3, and/or E4) of the adenoviral genome is removed such that the function of such region is eliminated or impaired.
  • a portion of the DNA sequence of a region of the adenoviral genome is removed such that one or more proteins normally encoded by such region are not expressed, or are expressed as a structure which eliminates or impairs the function of such proteins.
  • the determination of the amount of the DNA sequence of a region which is to be removed is determined by routine experimentation by one skilled in the art given the teachings herein. Preferably, all or substantially all of a coding region is removed.
  • At least open reading frame 6 (ORF 6) of the E4 DNA sequence is deleted. In another embodiment, at least open reading frames 3 and 6 of the E4 DNA sequence are deleted.
  • an adenoviral vector which includes an adenoviral 5' ITR; an adenoviral 3' ITR.* an adenoviral encapsidation signal; at least one DNA sequence encoding a protein or polypeptide of interest; and a promoter controlling the DNA sequence(s) encoding a protein(s) or polypeptide(s) of interest.
  • the adenoviral vector is free of all or a portion of the adenoviral El and all or a portion of E2 (preferably at least all or a portion of E2a) DNA sequences.
  • the adenoviral vector also is free of all or a portion of the adenoviral Elb DNA sequence.
  • Such vector is sometimes referred to as an adenoviral E1E2 " vector.
  • E2b DNA sequence is deleted. In yet another embodiment, all or a portion of the E2a and E2b DNA sequences are deleted.
  • an adenoviral vector which includes an adenoviral 5' ITR; an adenoviral 3' ITR; an adenoviral encapsidation signal; at least one DNA sequence encoding a protein or polypeptide of interest; and a promoter controlling the DNA sequence(s) encoding the protein(s) or polypeptide(s) of interest.
  • the adenoviral vector is free of all or a portion of the adenoviral El (including Ela and Elb), E2, and E4 DNA sequences.
  • Such vector is sometimes referred to as an adenoviral E1E2E4 ' vector.
  • such vector is free of at least open reading frame 6 of the E4 DNA sequence.
  • the vector is free of all or a portion of the E2a DNA sequence.
  • Such vector is sometimes referred to as an adenoviral El " E2a * E4 ' vector.
  • the vector is free of all or a portion of the E2b DNA sequence.
  • the adenoviral vectors hereinabove described also include at least a portion of the adenoviral E3 DNA sequence, operationally linked to a promoter which allows expression of E3 region genes in the transduced host cells, and which enables the adenovirus to retain some or all of its immune defense system.
  • the amount of the E3 DNA sequence included in the adenoviral vector is, in general, an amount of the E3 DNA sequence which will enable the adenoviral vector to evade or disable the host's immune response.
  • all of the genes of the E3 region are included in the vector, except the gene encoding the 11.6 Kda protein that causes cell lysis.
  • the above-mentioned adenoviral vectors do not contain the E3 region genes.
  • the vectors of the present invention in a preferred embodiment are adenoviral particles wherein the genetic material contained in the virus lacks the genetic material which is specified as having been deleted.
  • the genome of the adenoviral vector contains the early regions (El, E2, E3 and E4) and the major late regions to the extent necessary not to impair the function of the proteins encoded thereby, except for all or a portion of the regions which are specified as having been deleted from the genetic material of the adenovirus.
  • an adenoviral vector is defined as being free of all or a portion of El and E2
  • such vector would contain the E3 and E4 regions to the extent necessary not to disrupt or substantially impair the function of the protein encoded thereby. All other regions, including the major late regions and minor transcriptional units, would also be present to the extent necessary not to disrupt or substantially impair the function of the proteins encoded thereby.
  • the adenoviral vector may be derived from any known adenovirus serotype, including, but not limited to, Adenovirus 2, Adenovirus 3, and Adenovirus 5.
  • the adenoviral vector in general, also includes DNA encoding at least one therapeutic agent.
  • therapeutic is used in a generic sense and includes treating agents, prophylactic agents, and replacement agents.
  • DNA sequences encoding therapeutic agents which may be placed into the adenoviral vector include, but are not limited to, the CFTR gene,* genes encoding interferons such as Interferon-o., Interferon- ⁇ , and Interferon-7 ,* genes encoding interleukins such as IL-1, IL-l ⁇ , and Interleukins 2 through 14; genes encoding GM-CSF; genes encoding adenosine deaminase, or ADA; genes which encode cellular growth factors, such as ly phokines, which are growth factors for lymphocytes; genes encoding clotting factors such as Factor VIII and Factor IX; T-cell receptors,* the LDL receptor, ApoE, ApoCApoAI and other genes involved in cholesterol transport and metabolism; the alpha-1 antitrypsin (0.1AT) gene,* lung surfactant protein genes, such as the SP-A, SP-B, and SP-C genes,* the insulin gene, negative selective markers or "s
  • the DNA sequence (or transgene) which encodes the therapeutic agent may be genomic DNA or may be a cDNA sequence.
  • the DNA sequence also may be the native DNA sequence or an allelic variant thereof.
  • allelic variant as used herein means that the allelic variant is an alternative form of the native DNA sequence which may have a substitution, deletion, or addition of one or more nucleotides, which does not alter substantially the function of the encoded protein or polypeptide or fragment or derivative thereof.
  • the DNA sequence may further include a leader sequence or portion thereof, a secretory signal or portion thereof and/or may further include a trailer sequence or portion thereof.
  • Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or hetorologous promoters, such as the cytomegalovirus (CMV) promoter,* the Rous Sarcoma Virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters,* the albumin promoter; and the ApoAI promoter. It is to be understood, however, that the scope of the present invention is not to be limited to specific foreign genes or promoters.
  • CMV cytomegalovirus
  • RSV Rous Sarcoma Virus
  • Such vectors may be assembled by direct in vitro ligation from combinations of plasmids containing portions of modified or unmodified virus genome or plasmids and fragments derived directly from a linear adenoviral genome, such as the Adenovirus 5 genome (ATCC No. VR-5) or Adenovirus 5 derived viruses containing mutations or deletions as described herein.
  • a linear adenoviral genome such as the Adenovirus 5 genome (ATCC No. VR-5) or Adenovirus 5 derived viruses containing mutations or deletions as described herein.
  • the vectors can be assembled by homologous recombination, within a eukaryotic cell, between a plasmid clone containing a portion of the adenoviral genome (such as the Adenovirus 5 genome or the adenovirus 5 E3- mutant Ad dl327 (Thimmapaya, et al., Cell, Vol. 31, pg. 543 (1983)) with the desired modifications, and a second plasmid (such as, for example pAvS6, as shown in Figure 24) , containing the left adenoviral ITR, an El region deletion, and the desired trans gene.
  • a plasmid clone containing a portion of the adenoviral genome (such as the Adenovirus 5 genome or the adenovirus 5 E3- mutant Ad dl327 (Thimmapaya, et al., Cell, Vol. 31, pg. 543 (1983)) with the desired modifications
  • homologous recombination may be carried out between a plasmid clone and a fragment derived directly from a linear adenovirus (such as Adenovirus 5, or Ad dl327 or an Adenovirus 5 derived virus containing mutations or deletions as described herein) genome.
  • a linear adenovirus such as Adenovirus 5, or Ad dl327 or an Adenovirus 5 derived virus containing mutations or deletions as described herein
  • the vector then is transfected into a cell line capable of complementing the function of any essential genes deleted from the viral vector, in order to generate infectious viral particles.
  • the cell line in general is a cell line which is infectable and able to support adenovirus or adenoviral vector growth, provide for continued virus production in the presence of glucocorticoid hormones, and is responsive to glucocorticoid hormones (i.e., the cell line is capable of expressing a glucocorticoid hormone receptor) .
  • Cell lines which may be transfected with the essential adenoviral genes, and thus may be employed for generating the infectious adenoviral particles include, but are not limited to, the A549, KB, and HEp-2 cell lines.
  • the El region may be under the control of an inducible promoter.
  • inducible may include, but are not limited to, the mouse mammary tumor virus (MMTV) promoter (.Archer, et al . , Science. Vol. 255, pgs. 1573-1576 (March 20, 1992)),* the synthetic minimal glucocorticoid response element promoter GRE5 (Mader, et al . , Proc. Nat. Acad. Sci., Vol. 90, pgs.
  • MMTV mouse mammary tumor virus
  • the El region is under the control of an inducible promoter, and the E2a, E2b, and/or E4 regions are under the control of their native promoters.
  • the native promoters are transactivated by expression of the El region.
  • the cell line includes the entire adenoviral E4 region with its native promoter region, and the Ela region or the entire El region (including the Ela and Elb regions) under the control of a regulatable or inducible promoter, such as, for example, the mouse mammary tumor virus (or MMTV) promoter, which is a hormone inducible promoter, or other such promoters containing glucocorticoid responsive elements (GRE's) for transcriptional control.
  • the E4 DNA sequence also is expressed from a regulatable promoter, such as the MMTV promoter.
  • the El and E4 DNA sequences may be included in one expression vehicle, or may be included in separate expression vehicles.
  • the expression vehicles are plasmid vectors which integrate with the genome of the cell line.
  • the producer cell line includes a first plasmid which includes the Ela or the entire El DNA sequence under the control of an inducible promoter (such as the MMTV promoter) , and a second plasmid including the E4 DNA sequence under the control of its native promoter.
  • the producer cell line is transduced with the adenoviral vector which does not include the El and E4 DNA sequences.
  • the cells then are exposed to an inducing agent, (such as, for example, dexamethasone when an MMTV promoter is employed) , which activates the inducible promoter, such as the MMTV promoter, thereby initiating expression of the El DNA.
  • an inducing agent such as, for example, dexamethasone when an MMTV promoter is employed
  • the expression of the El DNA provides for transactivation of the E4 promoter, thereby providing for expression of the E4 DNA.
  • the expression of the El and E4 DNA thus enables the generation of infectious adenoviral particles.
  • the producer cell line includes a first plasmid which includes the Ela or the entire El DNA sequence under the control of an inducible promoter (such as the MMTV promoter or a promoter containing a glucocorticoid responsive element) , and a second plasmid including the E4 DNA sequence under the control of an inducible promoter (such as the MMTV promoter or a promoter containing a glucocorticoid responsive element) .
  • the producer cell line then is transduced with the adenoviral plasmid vector which does not include the El and E4 DNA sequences.
  • the cells then are exposed to the inducing agent (such as, for example, dexamethasone) , which activates the promoters (such as the MMTV promoters or promoters containing a glucocorticoid responsive element) controlling the El and E4 DNA sequences, thereby initiating expression of the El and E4 DNA sequences, whereby the expression of such sequences enables the generation of the infectious adenoviral particles.
  • the inducing agent such as, for example, dexamethasone
  • the promoters such as the MMTV promoters or promoters containing a glucocorticoid responsive element
  • the inducible or regulatable promoters controlling the El and E4 DNA sequences need not be identical.
  • an adenoviral vector wherein the Ela or entire El DNA sequence, and the E2a DNA sequence, or E2a and E2b DNA sequences, have been deleted, is transfected into a cell line capable of complementing the function of the Ela or entire El, and E2a or E2a and E2b DNA sequences, as well as any other essential genes deleted from the viral vector, in order to generate infectious viral particles.
  • the cell line includes the Ela or entire El region under the control of a promoter which may be its native promoter or a regulatable promoter, such as, for example, the mouse mammary tumor virus (or MMTV) promoter, or other such promoters containing glucocorticoid responsive elements (GRE's) for transcriptional control.
  • the cell line also includes the adenoviral E2a region or E2a and E2b regions under the control of its native promoter region, or a regulatable promoter such as those hereinabove described.
  • the Ela or entire El, and E2a or E2a and E2b DNA sequences may be included in one expression vehicle, or may be included in separate expression vehicles.
  • the producer cell line includes a first plasmid including the El DNA sequence under the control of a promoter containing a glucorticoid responsive element, and a second plasmid including the E2a DNA sequence under the control of the MMTV promoter.
  • the producer cell line is transduced with the adenoviral plasmid vector which does not include the El and E2a DNA sequences.
  • the cells then are exposed to a glucocorticoid hormone such as dexamethasone for activation of the El promoter and for activation of the MMTV promoter, thereby initiating expression of the El and E2a DNA sequences and enabling the generation of infectious adenoviral particles.
  • the producer cell line includes a first plasmid including the El DNA sequence under the control of an inducible promoter (such as the MMTV promoter or other promoters containing a glucocorticoid responsive element) , and a second plasmid including the E2a DNA sequence under the control of its native promoter.
  • an inducible promoter such as the MMTV promoter or other promoters containing a glucocorticoid responsive element
  • E2a DNA sequence under the control of its native promoter expression of the El and E2a DNA sequences is accomplished by the same approach as carried out with respect to the producer cell line including the El DNA sequence under the control of an inducible promoter, and the E4 DNA sequence under the control of its native promoter.
  • the producer cell line includes a first plasmid including the El DNA sequence under the control of the MMTV promoter, and a second plasmid including the E2a DNA sequence under the control of the MMTV promoter.
  • Expression of the El and E2a DNA sequences, leading to the generation of infectious adenoviral particles, is accomplished by exposing the cells to dexamethasone, thereby activating the MMTV promoters controlling the El and E2a DNA sequences.
  • an adenoviral vector wherein the El, E2a, and E4 DNA sequences have been deleted, is transfected into a cell line which includes the adenoviral El, E2a, and E4 regions. If the vector also includes a deletion of the E2b DNA sequence, the cell line also will include the E2b region.
  • Each of the El, E2a, and E4 regions may be under the control of their native promoters, or any or all of the El, E2a, and E4 regions may be under the control of an inducible or regulatory promoter, such as those hereinabove described.
  • the El, E2a, and B4 DNA sequences may be included in one expression vehicle, or the El, E2a, and E4 DNA sequences may each be included in separate expression vehicles, or two of the El, E2a, and E4 DNA sequences may be included in one expression vehicle, and the other of the El, E2a, and E4 DNA sequences may be included in another expression vehicle.
  • Expression of the El, E2a and E4 DNA sequences may be accomplished by employing approaches similar to those hereinabove described.
  • adenoviral vector particles of the present invention include the transduction of eukaryotic cells in vivo or in vitro as part of a gene therapy procedure, and the transduction of cells in vitro for the in vitro production of desired proteins or therapeutic agents.
  • the adenoviral vector particles are administered in vivo in an amount effective to provide a therapeutic effect in a host.
  • the vector may be administered in an amount of from 1 plaque forming unit to about 10 14 plaque forming units, preferably from about 10 6 plaque forming units to about lO 13 plaque forming units.
  • the host may be a mammalian host, including human or non-human primate hosts.
  • infectious adenoviral vector particles are administered to the lung when a disease or disorder of the lung (such as, for example, cystic fibrosis) is to be treated.
  • a disease or disorder of the lung such as, for example, cystic fibrosis
  • Such administration may be, for example, by aerosolized inhalation or brochoscopic instillation, or via intranasal or intratracheal instillation.
  • infectious adenoviral vector particles are administered systemically, such as, for example, by intravenous administration (such as, for example, portal vein injection or peripheral vein injection) , intramuscularadministration, intraperitoneal administration, intratracheal administration, or intranasal administration.
  • intravenous administration such as, for example, portal vein injection or peripheral vein injection
  • intramuscularadministration such as, for example, intraperitoneal administration, intratracheal administration, or intranasal administration.
  • the adenoviral vector particles may be administered in combination with a pharmaceutically acceptable carrier suitable for administration to a patient.
  • the carrier may be a liquid carrier (for example, a saline solution) , or a solid carrier, such as, for example, microcarrier beads.
  • Cells which may be infected by the infectious adenoviral particles include, but are not limited to, primary cells, such as primary nucleated blood cells, such as leukocytes, granulocytes, monocytes, macrophages, lymphocytes (including T-lymphocytes and B-lymphocytes) , totipotent stem cells, and tumor infiltrating lymphocytes (TIL cells) ,* bone marrow cells,* endothelial cells,* activated endothelial cells,* epithelial cells,* lung cells,* keratinocytes,* stem cells; hepatocytes, including hepatocyte precursor cells,* fibroblasts,* mesenchymal cells,* mesothelial cells,* parenchymal cells,* vascular smooth muscle cells,* brain cells and other neural cells,* gut enterocytes,* gut stem cells,* and myoblasts.
  • primary cells such as primary nucleated blood cells, such as leukocytes, granulocytes, mon
  • the infected cells are useful in the treatment of a variety of diseases including but not limited to adenosine deaminase deficiency, sickle cell anemia, thalassemia, hemophilia, diabetes, o.-antitrypsin deficiency, brain disorders such as Alzheimer's disease, phenylketonuria and other illnesses such as growth disorders and heart diseases, for example, those caused by alterations in the way cholesterol is metabolized and defects of the immune system.
  • diseases including but not limited to adenosine deaminase deficiency, sickle cell anemia, thalassemia, hemophilia, diabetes, o.-antitrypsin deficiency, brain disorders such as Alzheimer's disease, phenylketonuria and other illnesses such as growth disorders and heart diseases, for example, those caused by alterations in the way cholesterol is metabolized and defects of the immune system.
  • the adenoviral vector particles may be used to infect lung cells, and such adenoviral vector particles may include the CFTR gene, which is useful in the treatment of cystic fibrosis.
  • the adenoviral vector may include a gene(s) encoding a lung surfactant protein, such as SP-A, SP-B, or SP-C, whereby the adenoviral vector is employed to treat lung surfactant protein deficiency states.
  • the adenoviral vector particles may be used to infect liver cells, and such adenoviral vector particles may include gene(s) encoding clotting factor( ⁇ ), such as Factor VIII and Factor IX, which are useful in the treatment of hemophilia.
  • clotting factor( ⁇ ) such as Factor VIII and Factor IX
  • the adenoviral vector particles may be used to infect liver cells, and such adenoviral vector particles may include gene(s) encoding polypeptides or proteins which are useful in prevention and therapy of an acquired or an inherited defect in hepatocyte (liver) function. For example, they can be used to correct an inherited deficiency of the low density lipoprotein (LDL) receptor.
  • the adenoviral particles may be used to infect liver cells, whereby the adenoviral particles include a gene encoding a therapeutic agent employed to treat acquired infectious diseases, such as diseases resulting from viral infection.
  • the infectious adenoviral particles may be employed to treat viral hepatitis, particularly hepatitis B or non-A non-B hepatitis.
  • an infectious adenoviral particle containing a gene encoding an anti-sense gene could be employed to infect liver cells to inhibit viral replication.
  • the infectious adenoviral particle which includes a vector including a structural hepatitis gene in the reverse or opposite orientation, would be introduced into liver cells, resulting in production in the infected liver cells of an anti-sense gene capable of inactivating the hepatitis virus or its RNA transcripts.
  • the liver cells may be infected with an infectious adenoviral particle which includes a gene which encodes a protein, such as, for example, ⁇ -interferon, which may confer resistance to the hepatitis virus.
  • an adenoviral vector in accordance with the present invention may include a negative selective marker, or "suicide” gene, such as the Herpes Simplex Virus thymidine kinase (TK) gene.
  • a negative selective marker such as the Herpes Simplex Virus thymidine kinase (TK) gene.
  • TK Herpes Simplex Virus thymidine kinase
  • Such a vector may be employed in the treatment of tumors, including cancerous and non-malignant tumors, by administering the adenoviral vector to a patient, such as, for example, by direct injection of the adenoviral vector into the tumor, whereby the adenoviral vector transduces the tumor cells.
  • an interaction agent such as, for example, ganciclovir, is administered to the patient, whereby the transduced tumor cells are killed.
  • the viral particles which include at least one DNA sequence encoding a therapeutic agent
  • an adenoviral vector containing a DNA sequence encoding a therapeutic agent may be given to an animal which is deficient in such therapeutic agent.
  • the animal is evaluated for expression of such therapeutic agent. From the results of such a study, one then may determine how such adenoviral vectors may be administered to human patients for the treatment of the disease or disorder associated with the deficiency of the therapeutic agent.
  • the adenoviral vector particles may be employed to infect eukaryotic cells in vitro.
  • the eukaryotic cells may be those as hereinabove described.
  • Such eukaryotic cells then may be administered to a host as part of a gene therapy procedure in amounts effective to produce a therapeutic effect in a host.
  • the vector particles include a gene encoding a desired protein or therapeutic agent may be employed to infect a desired cell line in vitro, whereby the infected cells produce a desired protein or therapeutic agent in vitro.
  • the Adenovirus 5 genome was digested with Seal enzyme, separated on an agarose gel, and the 6,095 bp fragment comprising the left end of the virus genome was isolated.
  • the complete Adenovirus 5 genome is registered as Genbank accession #M73260, incorporated herein by reference, and the virus is available from the American Type Culture Collection, Rockville, Maryland, U.S.A., under accession number VR-5.
  • the Seal 6,095 bp fragment was digested further with Clal at bp 917 and Bglll at bp 3,328.
  • PCR Polymerase chain reaction
  • Ad5 bp 922-891 5' -03AGATCGATCACC7rCCGGTA ⁇ aAGGTTTGGCATAG-3'
  • This amplified DNA fragment (sometimes hereinafter referred to as Fragment A) then was digested with Xhol and Clal, which cleaves at the native Clal site (bp 917), and ligated to the Xhol and Clal sites of pSE280-E, thus reconstituting the 5' end of the El region beginning 8 bp upstream of the ATG codon.
  • Ad5 bp 3323-3360 5' -CATGAAGATCTGGAAGGTGCTGAGGTACGATGAGACC-3' 3' end
  • Ad5 bp 4090-4060 5' -CATGAAGATCTGGAAGGTGCTGAGGTACGATGAGACC-3' 3' end
  • This amplified DNA fragment (sometimes hereinafter referred to as Fragment B) was digested with Bglll, thereby cutting at the Adenovirus 5 Bglll site (bp 3,382) and EcoRI, and ligated to the Bglll and EcoRI sites of pSE280-AE to reconstruct the complete Ela and Elb region from Adenovirus 5 bp 552 through 4,090.
  • the resulting plasmid is referred to as pSE280-El ( Figure 1) .
  • the intact Ela/Elb region was excised from pSE280-El by cutting with EcoRI, blunting with Klenow and cutting with Sail.
  • the 3,567 bp fragment was purified from an agarose gel and ligated to the expression plasmid pMAMneo (Clonetech, Palo Alto, CA) , which was prepared similarly by cutting with Xhol at bp 3,405 of pMAMneo Luc ( Figure 2), blunting with Klenow, and cutting with Sail at bp 1,465.
  • the 8,399 bp fragment was gel purified, phosphatased, and ligated to the Ela/Elb fragment hereinabove described to form pMAMneo-El. ( Figure 3.)
  • Plasmid DNA was prepared and purified by banding in CsTFA. Circular plasmid DNA was linearized at the Xm I site within the ampicillin resistance gene of pMAMneo-El, and purified further by phenol/chloroform extraction and ethanol precipitation prior to use for transfection of cells.
  • the Adenovirus 5 genome was digested with BamHI and Spel, which cut at bp 21,562 and 27,080, respectively. Fragments were separated on an agarose gel and the 5,518 bp BamHI to Spel fragment was isolated. The 5,518 bp BamHI to Spel fragment was digested further with Smal, which cuts at bp 23,912. The resulting 2,350 bp BamHI to Smal fragment was purified from an agarose gel, and ligated into the superlinker shuttle plasmid pSE280, and digested with BamHI and Smal to form pSE280-E2 BamHI-Smal ( Figure 6) .
  • PCR then was performed to amplify Adenovirus 5 DNA from the Smal site at bp 23,912 through 24,730 contiguous with Nhel and EcoRI restriction sites.
  • the primers which were employed were as follows: 5' end, Ad5 bp 24,732-24,708:
  • Ad5 bp 23,912-23,934 5' -CACCCCGGGGAGGCGGCGGCGACGGGGACGGG-3'
  • the E2a region was excised from pSE280-E2a by cutting with BamHI and Nhel, arid recloned into the unique BamHI and Nhel sites of pSE280. ( Figure 6.) Subsequently, the E2a region was excised from this construction with Nhel and Sail in order to clone into the Nhel and Sail sites of the pMAMneo multiple cloning site in a 5' to 3' orientation, respectively. The resulting construct is pMAMneo E2a. ( Figure 6) .
  • Plasmid DNA was prepared and purified by banding in CsTFA. Circular plasmid DNA was linearized at the XmnI site within the ampicillin resistance gene of pMAMneo- E2a, and further purified by the phenol/chloroform extraction and ethanol precipitation prior to use for transfection of cells. Alternatively, the plasmid was linearized with Belli, which cuts in the reading frame of the neo R gene, thereby inactivating it. This form of the plasmid was used where co- transfection with an alternative selectable marker was required. Construction of plasmid including Adenovirus 5 ITR/E4 region.
  • the Adenovirus 5 genome was digested with Spel, which cuts at bp 27,082, and the two fragments separated on an agarose gel. The 8,853 bp fragment comprising the right end of Adenovirus 5 was isolated.
  • the 8,853 bp right end fragment was digested further with StuI at bp 31,956 and Eco47-III at bp 35,503. Fragments were separated on an agarose gel and the 3,547 bp StuI to Eco47-III fragment was isolated. This fragment was ligated to the superlinker shuttle plasmid pSE380 (Invitrogen) ( Figure 7) , digested with StuI and Eco47-IIl to construct PSE380-E4 ( Figure 8) .
  • PCR was performed to amplify Adenovirus 5 DNA from bp 35,499 through the right ITR ending at bp 35,935, and contiguous with a BamHI restriction site.
  • the PCR primers which were employed were as follows: 5' end, Ad5 bp 35,935-35,908: 5' -GTGGGATCC ⁇ TCAT ⁇ TAATATACCI ATTTTGGA-3' 3' end, Ad5 bp 35,503-35,536: 5' -ATAC ⁇ G ⁇ 3CT CC ⁇ C ⁇ G ⁇ i ⁇ CAGCCATAACAGTC-3'
  • a plasmid expressing all of the adenovirus 5 E4 open reading frames under the control of the glucocorticoid inducible GRE5 promoter, and containing a puromycin resistance gene under the control of the control of the SV40 promoter was constructed as follows.
  • the construct pSE380-E4 ( Figure 8) was cut with Eco47-3 and BamHI and ligated to a synthetic oligonucleotide of 33 bp, which includes the Eco47-3 site at Adenovirus 5 bp 35,503 through the E4 ORFl ATG at bp 35,522.
  • the resulting construct, pSE380E4-ATG ( Figure 9) contains all E4 open reading frames without a promoter.
  • the E4 ORF's were excised from pSE380E4/ATG as an Xhol to BamHI fragment and ligated to the Xhol and Bglll sites within the multiple cloning site of the expression shuttle plasmid pSE380GRE5/SV40Puro R , to generate pGRE5/E4Puro ( Figure 9) .
  • the expression shuttle plasmid pSE380GRE5/SV40Puro R was constructed from pSE380 ( Figure 7) , the GRE5 promoter excised from pGRE5/EBV ( Figure 4) , and a puromycin resistance gene.
  • this process involved the sequential introduction, by calcium phosphate precipitation, or other means of DNA delivery, of two plasmid constructions each with a different viral gene, into a single tissue culture cell.
  • the cells were transfected with a first construct and selected for expression of the associated drug resistance gene to establish stable integrants.
  • Individual cell clones were established and assayed for function of the introduced viral gene.
  • Appropriate candidate clones then were transfected with a second construct including a second viral gene and a second selectable marker. Transfected cells then were selected to establish stable integrants of the second construct, and cell clones were established. Cell clones were assayed for functional expression of both viral genes.
  • sequential transfections and selections were carried out with the following parental cell types:
  • Hep-2 (ATCC Accession No. CCL-23) ,* or
  • pMAMneo-E2a was digested with Belli to linearize the plasmid within the neo R reading frame and inactivate the gene.
  • the cell lines hereinabove described were co-transfected with the linear pMAMneo R -E2a and a selectable marker plasmid consisting of a eukaryotic expression cassette containing an SV40 promoter, a hygromyein resistance gene, and a polyadenylation signal cloned into the multiple cloning site of a pTZ18R ( Figure 10), at a 10:1 molar ratio.
  • E2a function was screened by complementation of the temperature- sensitive mutant Ad5tsl25 virus which contains a temperature- sensitive mutation in the E2a gene. (Van Der Vliet, et al., J. Virology. Vol. 15, pgs. 348-354 (1975)). Positive clones were identified and transfected with pMAMneoEl linearized with XmnI within the ampicillin resistance gene. The cells were selected for G418 resistance.
  • pMAMNeo-E2a was linearized with XmnI with the Amp R gene, introduced into cells by transfection, and cells were selected for stable integration of this plasmid by G418 selection. Clones expressing the E2a gene were identified and used for transfection with the 7 kb EcoRV to XmnI fragment from pGRE5-El ( Figure 5) , which contains the GRE5 promoted Ela/b region plus the hygromycin R gene. Cells were selected for hygromyein resistance and assayed for both Ela/b and E2a expression.
  • Circular plasmid pSE380-E4 was co-transfected into the cell lines hereinabove described with a hygromyein resistance plasmid as described above at a 10:1 molar ratio, 48 hours after transfection, the cells were placed under hygromyein selection and maintained in selection until drug resistant colonies arose. The clones then were isolated and screened for E4 function. Specifically, clones were tested for their ability to complement the Adenovirus 5 mutant dl 1011 (Bridge, et al . Virology. Vol. 193, pgs. 794-801 (1993)), which contains a deletion of all E4 open reading frames, and to produce infectious virus of the E4 genotype. Positive clones were identified and transfected with pMAMneoEl linearized with XmnI within the ampicillin resistance gene. The cells then were selected for G418 resistance. G418- re ⁇ istant colonies then were screened for El function.
  • clones were identified that inducibly express both viral genes. These cell lines are modified further by transfection with the pGRE5/E4/SV40Puro r plasmid ( Figure 9) and selection for puromycin resistant colonies. Cell clones are selected, expanded, and analyzed for inducible E4 function by assaying for dexamethasone-dependent complementation of the E4 deleted virus AdSdllOll. (Bridge, et al., Virology, Vol. 193, pg ⁇ . 794-801 (1993)). In another alternative, the E4 gene would be operationally linked to a tetracycline-inducible promoter ⁇ .
  • Adenovirus 5 genomic DNA was cut with BamHI (bp 21,562) and Spel (bp 27,082) restriction enzymes, and the 5,520 bp
  • pTZE2A was cut with BamHI (bp 8,391) and Smal (bp 2,350) restriction enzymes, corresponding to bp 21,562 and 23,912 of Adenovirus 5.
  • BamHI bp 8,391
  • Smal bp 2,350 restriction enzymes
  • the 6,041 bp and 2,350 bp BamHI/Smal fragments were isolated from an agarose gel.
  • the 2,350 bp BamHI/Smal fragment was digested with Dral restriction enzyme (bp 22,442 of Adenovirus 5) , and the 880 bp Dral to BamHI fragment was isolated from an agarose gel.
  • Such plasmid includes the Adenovirus 5 E2A region in which 1,470 bp of the E2A region, corresponding from the Dral site (bp 22,442) to the Smal site (bp 23,912) have been deleted. Subcloning of the Adenovirus 5 E3 region.
  • the Adenovirus 5 genome was digested with Spel (at bp 27,082).
  • the 8,853 bp fragment comprising the right end of Adenovirus 5 was isolated.
  • the 8,853 bp right end fragment of Adenovirus 5 was digested with StuI (bp 31,956) .
  • the 4,874 bp Spel/StuI fragment was isolated. This fragment was ligated to the plasmid pSE380 (Invitrogen) which was digested with StuI and Spel, in order to generate pSE380-E3+. ( Figure 12.)
  • the plasmid SE380-E3+ was digested with Spel and StuI, and the 4,874 bp fragment was ligated to the pSE380-ITR/E4 plasmid ( Figure 8) , which was digested with Spel and StuI, to generate pSE380-E3+E4+. ( Figure 13.)
  • the E3+/E4+ fragment then was excised as an 8,943 bp Spel to BamHI fragment from pSE380-E3+E4+, and reeloned into the Spel and BamHI sites of pTZ18R, previously modified to remove the Xbal site, to generate pTZE3+E4+. ( Figure 13.) Deletion within the E3 region.
  • the plasmid pTZE3+E4+ was digested with Xbal, in order to delete 1,878 bp of the E3 region between the Xbal site ⁇ at bp 28,592 and 30,470 of the Adenovirus 5 genome, to generate PTZE3 ⁇ 4+.
  • the plasmid pTZE3 " E4+ was digested with Spel and BamHI and ligated into the Spel and BamHI sites of the pSE280 plasmid to form pSE280-E3 ' E4+.
  • the pSE280-E3 " E4+ plasmid was dige ⁇ ted with BamHI, blunted, and recircularized to remove the BamHI site.
  • the E3 ⁇ 4+ containing fragment was removed by restriction digestion with Spel and Sail and ligated to the pSPORT-l ( Figure 15) (Gibco/BRL, Gaithersburg, MD) plasmid dige ⁇ ted with Spel and Sail, to generate pSPORTlE3 ' E4+.
  • Figure 16. Construction containing an E2A region deletion and a modified E3 region
  • the pSP0RTl/E2A-E3+E4+ ( Figure 16) may be modified by inserting an Xbal fragment containing a heterologous promotor, such as SV40, operationally linked to all or a part of the E3 region open reading frames into the unique Xbal site to generate pSPORTl/E2A-E3+*E4+.
  • a heterologous promotor such as SV40
  • the Adenoviru ⁇ 5 genome was digested with Clal (bp 917) and BamHI (bp 21,562) , and the 20,645 bp fragment was ligated to the plasmid pBR322E2A ' E3 ⁇ E4+ (which also was digested with Clal and BamHI), to generate pBRAd5E2A " E3 " E4+. ( Figure 18.) Intermediates for construction of a vector with an E4 deletion.
  • the plasmid pSE380-ITR/E4 was digested with BpU1102 (bp 33,129) and Eco47-3 (bp 35,503) to remove all E4 open reading frames, to generate pSE380-E4 " . ( Figure 19.)
  • a larger E4 region deletion is made which reduce ⁇ a 60 amino acid reading frame initiated from the E4 0RF1 ATG of pSE380E4 " to seven amino acids.
  • This preferred modification, referred to as pSE380E4 " (2) is constructed as shown in Figure 20.
  • pSE380E4 " is cut with Bpu 1102 (Adenovirus 5 bp 33,129) and Aflll (Adenovirus 5 bp 33,104), blunt ended by filing in 5' overhangs with Klenow, and religating to form pSE380E4 (2) .
  • This alternative E4 region deletion then is excised as a StuI to BamHI fragment and sub ⁇ tituted for the StuI to BamHI fragment in pSE380E3 + E4 + ( Figure 13) to generate pSE380E3 + E4 " (2) , a ⁇ ⁇ hown in Figure 20.
  • the Xbal region between Ad5 bp 28,592 and 30,470 in pSE380E3+E4 (2) ,* Figure 21 may be replaced with an Xbal fragment containing a heterologous promotor, such as SV40, operationally linked at some or all of the E3 region open reading frames, to create pSE380E3+*E4- (2) .
  • a heterologous promotor such as SV40
  • An Adenovirus 5 DNA fragment containing both the E3 region deletion and E4 region deletion in tandem is excised from pSE380E3 " E4"(2) by digestion with Spel (Adenovirus 5 bp 27,082) and Sail ( Figure 22). This fragment is substituted for the unique Spel to Sail fragment in pSPORTl/E2A " E3 ' E4 + ( Figure 16) to form pSPORTl/E2A ' E3 ' E4 ' (2) , as shown in Figure 22.
  • Virus Vector Production Production of a vector with the genotype E1+E2AE3E4+.
  • the plasmid pSPORT-lE2A " E3E4+ is digested with BamHI and Sail, and the fragment containing the E2AE3E4+ sequences is purified from an agarose gel.
  • the unique BamHI site correspond ⁇ to the BamHI site at bp 21,562 of Adenovirus 5.
  • the purified fragment is ligated in vi tro to the left side BamHI fragment of the Adenovirus 5 genome (21,562 bp) .
  • the ligation mixture then is transfected into an appropriate cell line capable of complementing the vector E2A deletion for production of an E1+E2E3 ⁇ 4+ Adenovirus 5 vector. ( Figure 23.)
  • the BamHI to Sail fragment may be isolated from pSPORTl/E2A-E3+*E4+ and used in the ligation with the Ad5 21,562 bp left end BamHI fragment to generate an Ad5 virus with the genotype E1+E2A-E3+*E4.
  • the DNA of the E1+E2 ⁇ 3 ⁇ 4+ Adenovirus 5 vector is purified and digested with Clal. This restriction enzyme cuts the Adenovirus 5 genome at bp 917.
  • the right side fragment is purified and co-transfected with pAvS6 (including a reporter gene or a trans-gene) into a double complementing cell line as capable of providing both El and E2A functions in trans.
  • pAvS6 ( Figure 24) is a shuttle plasmid which includes an adenoviral 5' ITR, and adenoviral encapsidation signal, an Ela enhancer sequence,* a promoter,* a tripartite leader sequence, a multiple cloning site for the insertion of foreign genes,- a poly A signal; and a DNA segment corresponding to the Adenovirus 5 genome which is no longer than from base 3329 to base 6246 of the genome.
  • pAvS ⁇ is described further in published PCT Application No. W094/23582, published October 27, 1994.
  • Thi ⁇ vector may be plaque purified further using a double complementing cell line, El + /E2a + .
  • a vector with the genotype E1E2A E3 ⁇ 4+ may be generated by ligating the left ITR through BamHI fragment of a pre-existing El " vector to the BamHI/Sall fragment of pSPORT-lE2A " E3 ' E4 " ( Figure 26) to generate directly an El " (reporter or trans-gene) E2AE3 ⁇ 4+ virus which will replicate when transfected into an appropriate complementing cell line.
  • Thi ⁇ approach require ⁇ that the reporter or tran ⁇ -gene doe ⁇ not contain a BamHI ⁇ ite.
  • a vector with the genotype El " E2a " E3'E4 + was generated using the adenoviral pla ⁇ mid vector pAvS6nLacZ (described in PCT application No. W095/09654) , which contains a nuclear localizing beta-galactosidase cDNA.
  • the resulting vector, Av3nLacZ ( Figure 28) was generated by homologous recombination as outlined in Figure 25.
  • the vector Av3nLacZ can be used as a starting material for the production of other vectors.
  • Av3nLacZ contain ⁇ two Clal ⁇ ite ⁇ , one within the lacZ coding region and one at the 3' end of lacZ within the polyadenylation signal. ( Figure 28) .
  • Figure 28 By digesting Av3nLacZ with Clal and recombining with pAvS6/transgene as described in Figure 25, other Av3-type vectors are made. This approach provides advantages in that no El + vector backbone is present in the recombination, and new recombinant vectors can be ⁇ elected a ⁇ B-gal negative when X-gal is included in the agar overlay during plaque purification.
  • DNA from the virus E1+E2A-E3+*E4+ is digested with Clal and the right side fragment used to co-tran ⁇ fect with pAVS6/tran ⁇ gene into E1/E2A expressing cells to generate, by homologou ⁇ recombination, a vector with the genotype E1-E2A-E3+*E4+.
  • the left end BamHI fragment from a pre-exi ⁇ ting El- vector may be ligated directly to the BamHI/Sail fragment from a pSPORTl/E2A-E3+*E4+ and tran ⁇ fected into E1/E2A expressing cells to generate a vector with the genotype E1-, transgene, E2A-E3+*E4+.
  • the in vi tro ligation mixture is transfected into an E4+ cell line to produce an Adenovirus 5 E1+E4 " vector.
  • pSE380E3 + E4 " (2) is used to generate an El + E3 + E4'(2) virus with the E4 deletion in the same manner as outlined in Figure 29.
  • pSE380E3E4 " (2) is used in the same manner to generate an E1 + E3 " E4 " (2) adenovirus. Production of a vector with genotype E1E4 " .
  • the DNA of the Adenovirus 5 E1+E4 genome is purified and digested with Clal restriction enzyme.
  • the right ⁇ ide fragment i ⁇ purified and co-transfected with pAvS6 (containing a reporter or trans-gene) into a double complementing cell line expressing El and E4.
  • Homologous recombination between the E4 " viral fragment and pAvS6 will result in a vector with the genotype El " (reporter or trans ⁇ gene) E2A + E3+E4 " .
  • the vector ( Figure 30) is plaque purified using a double complementing cell line.
  • the Spel/BamHI fragment from SE380-E3+E4 " is ligated directly to the left end fragment of a pre-existing El " vector digested with Spel to generate an El " (reporter or trans-gene) ) E4 " vector. ( Figure 31.)
  • This approach requires that the reporter or trans-gene does not contain an Spel site.
  • tran ⁇ gene-containing vectors of the types El ' (tran ⁇ gene) E3 + E4 ' (2) or El " (tran ⁇ gene) E3 " E4 " (2) are generated.
  • Adenoviru ⁇ vector ⁇ with deletion ⁇ in 3 essential genes (El, E2A, E4) , referred to as triple deletion vector ⁇ or AV4 generation vector ⁇ are con ⁇ tructed as follows. Using Av3nLacZ as a starting material, AV4nLacZ is generated by either homologous recombination or in vitro ligation. Using AV4nLacZ as a ⁇ tarting material, any other tran ⁇ gene containing Av4 type vector can be con ⁇ tructed.
  • the unique BamHI to Sail fragment from pSPORTl/E2A " E3 " E4 " (2) i ⁇ i ⁇ olated and co-tran ⁇ fected with the left end fragment of Av3nLacZ cut at the unique Srfl restriction site (bp 27,098 of AV3nLacZ (Fig ⁇ . 28, 32) into a triple complementing cell line (as described in Example 1 hereinabove) expressing El, E2A, and E4 viral genes.
  • the unique Srfl to Sail fragment from pSPORTl/E2A " E3 " E4 (2) may be i ⁇ olated and directly ligated to the left end fragment of AV3nLacZ cut with Srfl at bp 27,098 to yield a vector with the genotype El " , nLacZ, E2A", E3 " , E4 " .
  • This ligation mixture may then be transfected into the triple complementing cell line (El + , E2A + , E4 + ) to produce AV4nLacZ (Fig. 33) .
  • AV4 vectors containing transgenes other than nLacZ are constructed.
  • Purified DNA from Av4nLacZ is digested with Clal (See Fig. 34) , and the right end viral fragment is purified.
  • This fragment is co- transfected with the desired pAVS6/transgene construct into triple complementing cells (El + , E2A + , E4 + ) and by homologous recombination to yield an AV4 type vector with the genotype El " , transgene, E2A “ , E3 " , E4 " (Fig. 34) .
  • a unique BamHI/Sall fragment from pSPORTl/E2A-E3+*E4- (2) may be isolated and co-transfected with the left end fragment of AV3nLacZ cut at the unique Srfl restriction site into cells expressing El, E2A, and E4 in order to generate, by homologous recombination, a vector with the genotype El-,nLacZ,E2A-E3+*E4 (2) or AV4nLacZE3+*.
  • the unique Srfl to Sail fragment from pSPORTl/E2A-E3+*E4- (2) may be isolated and used to ligate directly to the left end fragment of AV3nLacZ cut with Srfl.
  • the ligation product is then transfected into cells expres ⁇ ing El, E2A, and E4 to generate a vector with the genotype El-,nLacZ,E2A-E3+*E4- (2) , or AV4nLacZE3+*.
  • AV4 vector ⁇ containing a tran ⁇ gene other than nLacZ and containing the modified E3+* region can be generated in a fashion analogous to that shown in Figure 34.
  • AV4nLacZE3+* is cut with Clal and co- transfected with pAV6/transgene into cells expressing El, E2A, and E4 to generate a vector with the genotype El-, transgene, E2A-E3+*E4- (2) .
  • the normal human CFTR cDNA sequence (nucleotides 75 to 4,725; for numbering see Gen Bank accession number M28668) was removed from plasmid pBQ4-7 ( Figure 35) by PstI digestion followed by blunting of the CFTR cDNA ends with T4 polymera ⁇ e (pBQ4-7 wa ⁇ provided by L.-C.T ⁇ ui, the Ho ⁇ pital for Sick Children, Toronto, Canada.) Thi ⁇ CFTR cDNA wa ⁇ then in ⁇ erted into the linearized pAvS6 pla ⁇ mid ⁇ o a ⁇ to create an operational linkage between the RSV promoter and the 5' end of the coding ⁇ equence of the CFTR cDNA.
  • the re ⁇ ulting plasmid pAvS6 CFTR ( Figure 35) was linearized by digestion with Kpnl and cotransfected into El/E2a, E1/E4 or El/E2a/E4 expre ⁇ ing cell ⁇ along with the large Clal fragment of Av3LacZ for El"/ E2 " , AvSLacZ for E1/E2/E4 " , or the large Clal fragment from Av4nLacZ, re ⁇ pectively, as described above.
  • iEl/E2a cells cotransfected as described above were then overlaid with agent and cultured in a humidified atmosphere containing 5% C0 2 at 37°C until formation of virus plaques.
  • Plaques were picked and the recombinant adenovirus vectors further plaque-purified, amplified and titered as previously described (Rosenfeld, et al., Cell. Vol. 68, pgs. 143-155
  • Adenoviral vectors are evaluated for deletion of Ad genes El/B2a, E1/E4 or E1/E2/E4 and inclusion of part or all of the normal human CFTR cDNA as previously described
  • Example 5 Demonstration that El/E2a-deleted vectors have reduced expres ⁇ ion of adenoviral late gene product ⁇ .
  • This example demonstrate ⁇ that third generation vector ⁇ which harbor deletion ⁇ in all or part of El/E2a with or without deletions of E3 region sequences have reduced potential for elicitation of a host immune response.
  • the expres ⁇ ion of hexon gene was measured by metabolic labeling. Further, this expression was compared among a first generation vector (e.g., E1/E3 deleted), a second generation vector (e.g., El deleted containing a temperature sensitive mutation in the E2a gene (Englehardt, et al., Proc. Nat. Acad. Sci.. Vol. 91, pgs. 6196-6200 (1994)) and the third generation vector described above.
  • a first generation vector e.g., E1/E3 deleted
  • a second generation vector e.g., El deleted containing a temperature sensitive mutation in the E2a gene
  • A549 cells were infected with AvlLacZ4 (50-500 iu/cell) Av2Lucl, which is an El deleted vector containing a temperature sensitive mutation in the E2a gene, and a luciferase gene. (50-500 iu/cell) . or Av3LacZl (500-3000 in/cell) and cultured in a humidified atmosphere with 5% Co 2 at 37°C.
  • AvlLacZ4 50-500 iu/cell
  • Av2Lucl an El deleted vector containing a temperature sensitive mutation in the E2a gene, and a luciferase gene.
  • Av3LacZl 500-3000 in/cell
  • iEl/E2a cells or 293 cells were infected with AvlLacZ4, Av2Lucl or Av3LacZl at an approximate multiplicity of infection (M.O.I.) of 10 infectious units (iu) per cell.
  • Media was then changed to methionine-free DMEM containing 35 S-Met (Mittereder, et al., 1994) and cultured for an additional 24 hours.
  • Metabolically labelled cells were then washed in phosphate buffered ⁇ aline, lysed and then scraped into 800 ⁇ l of Ripa buffer containing antiproteases (PMSF, leupeptide and proteinase) .
  • Example 6 Demonstration that El/E2a-deleted vectors have reduced replication of the adenoviral DNA genome.
  • third generation (El " /E2a) vectors with or without deletions of the E3 region sequences have improved characteristics with respect to residual viral DNA replication.
  • metabolic labeling of vector infected cells with 32 P was conducted. These experiments were carried out in a manner analogous to that described in Example 5 above.

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Abstract

L'invention concerne un vecteur adénoviral où le génome adénoviral a été modifié pour diminuer chez l'hôte l'inflammation et la réponse immunitaire vis-à-vis du vecteur. Dans une forme d'exécution, le vecteur adénoviral comprend une séquence adénovirale 5'-ITR, une séquence adénovirale 3'-ITR, un signal d'encapsidation adénoviral, au moins une séquence d'ADN codant pour une ou des protéines ou pour un ou des polypeptides utiles, et un promoteur contrôlant la ou les séquences d'ADN codant pour la ou les protéines ou le ou les polypeptides utiles. Le vecteur adénoviral est exempt de la totalité ou d'une partie de chacune des séquences E1 et E4 d'ADN adénoviral, ou il est exempt d'une partie de chacune des séquences E1 ou E2 d'ADN adénoviral, ou il est exempt d'une partie ou de la totalité de chacune des séquences E1, E2 et E4 d'ADN adénoviral. On décrit et on revendique également une cellule productrice pour produire des particules de vecteur adénoviral, où la cellule productrice comprend une séquence E1 d'ADN adénoviral et une séquence E4 d'ADN adénoviral ou elle comprend une séquence E1 d'ADN adénoviral et une séquence E2a d'ADN adénoviral, ou les séquences E1, E2a et E4 d'ADN adénoviral.
EP95943705A 1994-12-12 1995-12-07 Vecteurs adenoviraux ameliores et cellules productrices Withdrawn EP0797454A4 (fr)

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KR20170036085A (ko) 2014-07-31 2017-03-31 더 보드 오브 리젠츠 오브 더 유니버시티 오브 오클라호마 포유동물 rpe65의 높은 아소머로하이드롤라아제 활성 돌연변이
EP3390428B1 (fr) 2016-02-23 2019-09-25 Salk Institute for Biological Studies Dosage à haut débit pour mesurer la cinétique de réplication d'un adénovirus
CA3013639A1 (fr) 2016-02-23 2017-08-31 Salk Institute For Biological Studies Expression de genes exogenes dans un adenovirus therapeutique pour un effet minimal sur la cinetique virale
JP2019536468A (ja) 2016-12-12 2019-12-19 ソーク インスティテュート フォー バイオロジカル スタディーズ 腫瘍を標的にする合成アデノウイルスおよびその使用
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