EP1073758A1 - Vecteurs retroviraux incluant des proteines d'escorte a enveloppe modifiee - Google Patents

Vecteurs retroviraux incluant des proteines d'escorte a enveloppe modifiee

Info

Publication number
EP1073758A1
EP1073758A1 EP99914700A EP99914700A EP1073758A1 EP 1073758 A1 EP1073758 A1 EP 1073758A1 EP 99914700 A EP99914700 A EP 99914700A EP 99914700 A EP99914700 A EP 99914700A EP 1073758 A1 EP1073758 A1 EP 1073758A1
Authority
EP
European Patent Office
Prior art keywords
protein
retroviral
modified
envelope protein
vector
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.)
Ceased
Application number
EP99914700A
Other languages
German (de)
English (en)
Inventor
Frederick L. Hall
Erlinda Maria Gordon
W. French Anderson
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.)
University of Southern California USC
Original Assignee
University of Southern California USC
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 University of Southern California USC filed Critical University of Southern California USC
Publication of EP1073758A1 publication Critical patent/EP1073758A1/fr
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • A61P13/00—Drugs for disorders of the urinary system
    • 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
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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
    • A61P25/26—Psychostimulants, e.g. nicotine, cocaine
    • 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
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13045—Special targeting system for viral 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
    • C12N2810/405—Vectors comprising RGD peptide
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/55—Vectors comprising as targeting moiety peptide derived from defined protein from bacteria
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/851—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from growth factors; from growth regulators
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/857—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from blood coagulation or fibrinolysis factors
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/859—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from immunoglobulins

Definitions

  • This invention relates to retroviral vectors which are "targeted” for binding to a desired target molecule. More particularly, this invention relates to retroviral vectors having a first envelope protein and at least one modified envelope protein.
  • the first envelope protein includes a surface protein including a receptor binding region, a hypervariable polyproline region, and a body portion.
  • the at least one modified envelope protein is a modified retroviral envelope protein in which at least 90% of the amino acid residues of the receptor binding region of the envelope protein are removed and replaced with a non-retroviral peptide.
  • the non-retroviral peptide may be a ligand which binds to a desired target molecule.
  • target molecule means a molecule which is capable of being bound by the ligand. Such molecules include, but are not limited to, cellular receptors, extracellular components such as extracellular matrix components, and antibodies.
  • Retroviral vector particles are useful agents for transducing polynucleotides into cells, such as eukaryotic cells.
  • retroviral vector particles have been used for introducing polynucleotides into cells for gene therapy purposes.
  • cells are obtained from a patient, and retroviral vector particles are used to introduce a desired polynucleotide into the cells, and such modified or engineered cells are returned to the patient for a therapeutic purpose.
  • retroviral vector particles may be administered to the patient in vivo, whereby the retroviral vector particles transduce cells of the patient in vivo.
  • retroviral vector particle infection In many gene therapy protocols, it would be desirable to target retroviral vector particle infection to a specific population of cells either in vivo or in vitro. In such circumstances, the broad host range of typical retroviruses present a significant problem.
  • a key determinant of viral host range is the "envelope" or "env” protein (encoded by the env gene) which is involved in binding to receptors on the surface of susceptible cells.
  • Retroviral vectors have been made which have modified envelopes; however, such vectors in general are less infective than wild-type retroviral vectors or retroviral vectors including foreign genes, but which have unmodified envelopes.
  • the present invention provides "modified env proteins" that permit the incorporation, expression and assembly of large poiypeptides within the basic framework (i.e. N-terminal signal peptide, N-terminus, surface (SU) C-terminus and membrane spanning transmembrane (TM) domains) of the env protein of a virus, for example a retrovirus. These modified env proteins are devoid of much of the receptor binding domains.
  • Escort protein necessarily requires co-expression with a wild type env to gain infectivity.
  • the "escort protein” provides the gain of function; i.e. targeting motif that directs or escorts the virus to the specific target cell or target ligand, such as IgG or exposed collagen or ECM.
  • a "retroviral vector particle” is an infectious virion derived from a retrovirus.
  • a "retroviral vector plasmid vector” is a non-infectious plasmid comprising retroviral DNA, wherein said plasmid is capable of use as a vector for transfection of a target cell
  • Retroviral DNA is DNA transcribed from retroviral RNA by reverse transcriptase.
  • the present invention provides a retroviral vector which includes a first envelope protein and at least one modified envelope protein.
  • the first envelope protein includes a surface protein which includes a receptor binding region, a hypervariable polyproline region, and a body portion.
  • Such an envelope protein may be an unmodified, or wild-type, envelope protein, or may be modified at the N-terminus without removing any portion of the receptor-binding domain, such as by insertion of a small peptide or ligand.
  • the at least one modified envelope protein has been modified such that at least a major portion of the receptor binding region of such envelope protein has been removed and - 3 -
  • a non-retroviral protein or peptide such as a ligand that binds to a desired target molecule.
  • Figure 1 shows the results of an ELISA assay in which collagen-coated wells were contacted with the retroviral vectors WT-CEE, BS-CEE.CEE, BN-CEE.CEE, and BA- CEE.CEE; and
  • Figure 2 shows the results of an ELISA assay in which Ig G coated wells were contacted with retroviral vectors including an envelope "escort" protein which includes Protein A.
  • modified retroviral envelope protein as will be described further hereinbelow.
  • modified retroviral envelope prior to modification, includes a surface protein which includes a receptor binding region, a hypervariable polyproline region, and a body portion.
  • the modified retroviral envelope protein has been modified such that at least 90% of the amino acid residues of the receptor binding region of the surface protein have been removed and replaced with a non-retroviral protein or peptide.
  • modified retroviral envelope protein in general may be included in a retroviral vector.
  • the retroviral vector includes the modified retroviral envelope protein as well as a retroviral envelope protein in which the receptor binding region, the hypervariable polyproline region, and the body portion have not been modified.
  • a retroviral vector including a first retroviral envelope protein and at least one modified retroviral envelope protein.
  • the first retroviral envelope protein includes a surface protein.
  • the surface protein includes (i) a receptor binding region; (ii) a hypervariable polyproline, or "hinge” region, and (iii) a body portion.
  • the modified retroviral envelope protein prior to modification, includes a surface protein which includes (i) a receptor binding region; (ii) a hypervariable polyproline, or "hinge” region; and (iii) a body portion.
  • the modified retroviral envelope protein has been modified such that at least 90% of the amino acid residues of the receptor binding region of the surface protein of the modified retroviral envelope protein have been removed and replaced with a non-retroviral protein or peptide, such as for example, a ligand which binds to a desired target molecule. In one embodiment, at least 92% of the amino acid residues of the receptor binding region of the surface protein of the modified retroviral envelope protein have been removed and replaced with a non-retroviral protein or peptide, such as a ligand that binds to a desired target molecule. In another embodiment, all of the amino acid residues of the receptor binding region of the surface protein of the modified retroviral envelope protein have been removed and replaced with a non-retroviral protein or peptide.
  • At least 90% of the amino acid residues of the receptor binding region of the surface protein of the modified retroviral envelope protein have been removed and replaced with a non-retroviral protein or peptide, and at least a portion of the amino acid residues of the hypervariable polyproline region of the surface protein of the modified retroviral envelope protein have been removed and replaced with a non-retroviral protein or peptide. In one embodiment, all of the amino acid residues of the hypervariable polyproline region of the modified retroviral envelope protein have been removed.
  • the receptor binding region(s) of the modified retroviral envelope protein(s), prior to modification thereof, has (have) the sequence (SEQ ID NO:1).
  • amino acid residues 19 through 229 of (SEQ ID NO:1) have been removed and replaced with a non-retroviral protein or peptide.
  • amino acid residues 19 through 229 of (SEQ ID NO:1 ) and at least a portion of the amino acid residues of the hypervariable polyproline region of the surface protein of the modified retroviral envelope protein(s) have been removed and replaced with a non- retroviral protein or peptide.
  • retroviral envelope protein(s) include a surface (SU) domain, or surface protein, and a transmembrane (TM) domain or protein.
  • the surface protein includes, in an N-terminal to C-terminal direction, the following regions: (i) a receptor binding region; (ii) a hypervariable polyproline region; and (iii) a body portion, which is associated with the transmembrane domain.
  • the first retroviral envelope protein includes the surface domain and the transmembrane domain. In general, such envelope protein is free of non-retroviral peptides.
  • the first retroviral envelope protein maintains wild-type infectivity.
  • the first retroviral envelope protein in one embodiment, may include regions of different tropisms.
  • the first retroviral envelope protein may include a surface protein which includes (i) an ecotropic receptor binding region; (ii) an amphotropic hypervariable polyproline region; and (iii) an ecotropic body By "amphotropic" is meant - 5 -
  • ecotropic capable of infecting rodent cells only.
  • the modified retroviral envelope protein(s) is (are) a retroviral envelope protein(s) which is (are) modified such that at least 90% of the amino acid residues of the receptor binding region of the surface protein have been removed and replaced with a non-retroviral protein or peptide.
  • Shown in (SEQ ID NO:1 ) is the receptor binding region of the ecotropic envelope of Moloney Murine Leukemia Virus.
  • a retroviral vector that includes a first retroviral envelope protein which maintains wild-type infectivity and retains a receptor binding region, an unmodified hypervariable polyproline region, and an unmodified body portion; and at least one modified retroviral envelope protein in which at least 90% of the amino acid residues of the receptor binding region of the surface protein have been removed and replaced with a non-retroviral protein or peptide, the modified retroviral envelope protein(s) serves as an "escort-protein" which provides one or more additional functions to the retroviral vector, such as, for example, "targeting" the retroviral vector to a desired target molecule.
  • Such retroviral vectors while possessing such additional functions, retain the infectivity of wild- type retroviruses.
  • the modified retroviral envelope protein(s), prior to the modification of at least the receptor binding region to include the non-retroviral protein or peptide may be an envelope which includes regions of different tropisms.
  • the modified retroviral envelope protein(s) may be a Moloney Murine Leukemia Virus envelope protein(s) which includes a surface protein (also known as gp 70 protein) having an ecotropic portion and an amphotropic portion and/or xenotropic portion
  • the modified retroviral envelope protein prior to modification thereof, has a gp 70 protein which includes: (i) an ecotropic receptor binding region, i.e., (SEQ ID NO:1 ); (ii) an amphotropic hypervariable polyproline region, (SEQ ID NO:2); and (iii) an ecotropic body portion. At least 90% of the amino acid residues of the ecotropic receptor binding region (SEQ ID NO:1 ) have been removed and replaced as hereinabove described, with a non-retroviral protein or peptide. In a further embodiment, at least a portion of the amphotropic hypervariable polyproline region (SEQ ID NO:2) have been removed as well.
  • amino acid residues 1 through 35 of (SEQ ID NO:2) have been removed.
  • amino acid residues 1 through 48 of (SEQ ID NO:2) have been removed.
  • all 60 amino acid residues of (SEQ ID NO:2) have been removed.
  • the retroviral vector particle includes a first retroviral envelope protein and a modified retroviral envelope protein.
  • the first retroviral envelope protein includes a surface protein including a receptor binding region, a hypervariable polyproline region, and a body portion as hereinabove described.
  • the non-retroviral protein or peptide is a ligand which binds to a desired target molecule.
  • the ligand includes a binding region which binds to a receptor located on a desired cell type.
  • ligands include, but are not limited to, antibodies and fragments thereof, including single-chain antibodies, monoclonal antibodies, and poiyclonal antibodies.
  • Such antibodies include, but are not limited to, antibodies and fragments or portions thereof which bind to erb-B2, such as, for example, e23 antibody; antibodies which bind to receptors such as, for example, the CD4 receptor on T-cells; antibodies which bind to the transferrin receptor; antibodies directed against human leukocyte antigen (HLA); antibodies to carcinoembryonic antigen; antibodies to placental alkaline phosphatase found on testicular and ovarian cancer cells; antibodies to high molecular weight melanoma- associated antigen; antibodies to polymorphic epithelial mucin found on ovarian cancer cells; antibodies to ⁇ -human chorionic gonadotropin; antibodies to CD20 antigen of B- lymphoma cells; antibodies to alphafetoprotein; antibodies to prostate specific antigen; OKT-3 antibody, which binds to CD3 T-lymphocyte surface antigen; antibodies which bind to B-lymphocyte surface antigen; antibodies which bind to EGFR (c-erb-B
  • cytokines include, but are not limited to, interleukins, including interleukin-l ⁇ , interleukin-l ⁇ , , and Interleukins 2 through 14; growth factors such as epithelial growth factor (EGF), TGF-OC, TGF- ⁇ , , fibroblast growth factor (FGF), keratinocyte growth factor (KGF), PDGF-A, PDGF- B, PD-ECGF, IGF-I, IGF-II, and nerve growth factor (NGF), which binds to the NGF receptor of neural cells; colony stimulating factors such as GM-CSF, G-CSF, and M-CSF, leukemic inhibitory factor (LIF); interferons such as interferon- ⁇ , interferon- ⁇ , and interferon- ⁇ , inhibin A; inhibin B; chemotactic factors; ⁇ -type intercrine cytokines; and ⁇ -type intercrine
  • Still other ligands which may be employed include, but are not limited to, vascular endothelial growth factor, or VEGF, melanoma stimulating hormone, which binds to the MSH receptor on melanoma cells; the polypeptide FLA16, which has the sequence Cys- Gln-Ala-Gly-Thr-Phe-Ala-Leu-Arg-Gly-Asp-Asn-Pro-Gln-Gly-Cys,(SEQ. ID. NO.
  • polypeptide having the structure Gly-Glu-Arg-Gly-Asp-Gly-Ser-Phe-Phe-Ala-Phe-Arg- Ser-Pro-Phe, (SEQ. ID. NO.
  • erythropoietin which binds to the erythropoietin receptor
  • adherins selectins
  • CD34 which binds to the CD34 receptor of hematopoietic stem cells
  • CD33 which binds to premyeloblastic leukemia cells
  • stem cell factor stem cell factor
  • asialoglycoproteins including asialoorosomucoid, asialofetuin, and alpha-1 acid glycoprotein, which binds to the asialoglycoprotein receptor of liver cells
  • insulin glucagon
  • gastrin poiypeptides which bind to receptors on hematopoietic stem cells
  • C-kit ligand tumor necrosis factors (or TNF's) such as, for example, TNF-alpha and TNF-beta
  • ApoB which binds to the LDL receptor of liver cells
  • alpha-2 tumor necrosis factors
  • ApoE apolipoprotein E
  • the ligand is a single chain antibody.
  • the ligand includes a binding region which binds to an extracellular matrix component.
  • extracellular matrix component means a molecule that occupies the extracellular spaces of tissues.
  • extracellular matrix components include, but are not limited to, collagen (including collagen Type I and collagen Type IV), laminin, fibronectin, elastin, glycosaminoglycans, proteoglycans, and sequences which bind to fibronectin, such as arginine-glycine-aspartic acid, or RGD, sequences.
  • Binding regions which bind to an extracellular matrix component, and which may be included in a targeting polypeptide include, but are not limited to, polypeptide domains which are functional domains within von Willebrand Factor or derivatives thereof, wherein such polypeptide domains bind to collagen.
  • the binding region is a polypeptide having the following structural formula: Trp-Arg-Glu-Pro-Ser-Phe-Met-Ala- Leu-Ser. (SEQ. ID. NO. 7)
  • binding regions which bind to an extracellular matrix component, and which may be included in the second retroviral envelope include, but are not limited to, the arginine- glycine-aspartic acid, or RGD, sequences, which binds fibronectin, and a polypeptide having the sequence Gly-Gly-Trp-Ser-His-Trp, (SEQ. ID. NO. 8) which also binds to fibronectin.
  • the ligand may further include linker sequences of one or more amino acid residues, placed at the N-terminal and/or C-terminal of the binding region, whereby such linkers increase rotational flexibility and/or minimize steric hindrance of the modified envelope polypeptide.
  • the ligand is a peptide or protein which binds to an antibody.
  • proteins or peptides include, but are not limited to, the Ig G-binding domain of Protein A, synthetic Ig G-binding domains, such as Protein ZZ, and Protein G.
  • a modified polynucleotide encoding a modified retroviral envelope polypeptide (i.e., the modified retroviral envelope or "escort" protein hereinabove described).
  • the retroviral envelope polypeptide includes a receptor binding region.
  • polynucleotide encoding at least 90% of the amino acid residues of the receptor binding region has been removed and replaced with a polynucleotide encoding a non-retroviral protein or peptide, as hereinabove described, such as, for example, a ligand which binds to a desired target molecule.
  • the polynucleotide encoding the receptor binding region encodes the sequence of (SEQ ID NO:1 ).
  • a polynucleotide including the codons encoding amino acid residues 19 through 229 of (SEQ ID NO:1 ) has been removed and replaced with the polynucleotide encoding the ligand.
  • a polynucleotide encoding at least a portion of the hypervariable polyproline region also has been removed as well.
  • the hypervariable polyproline region has the sequence (SEQ ID NO:2).
  • the receptor binding region having the sequence (SEQ ID NO:1 ) is encoded by the polynucleotide having (SEQ ID NO:3) or a degenerative derivative or analogue thereof.
  • the hypervariable polyproline region having the sequence (SEQ ID NO:2) is encoded by the polynucleotide having (SEQ ID NO:4) or a degenerative derivative or analogue thereof.
  • derivative or analogue thereof as used herein means that the polynucleotides encoding the poiypeptides (SEQ ID NO:1 ) and (SEQ ID NO:2) may have sequences different from the polynucleotides (SEQ ID NO:3) and SEQ ID NO:4), yet encode the same polypeptide. Such differences in polynucleotide sequences may, for example, be due to the degeneracy of the genetic code.
  • (SEQ ID NO:2) or (SEQ ID NO:4) may be modified such that one or more codons encode different amino acid residues than the unmodified sequences. Such modifications may facilitate the insertion of the polynucleotide encoding the ligand.
  • a first expression plasmid may be constructed which includes a polynucleotide encoding the unmodified envelope protein.
  • the plasmid then is engineered such that a polynucleotide encoding at least 90% of the amino acid residues of the receptor binding region, and which, in some embodiments, also may encode at least a portion of the hypervariable polyproline region, has been removed, whereby such polynucleotide has been replaced with a polynucleotide encoding the ligand.
  • the polynucleotide encoding the ligand may be contained in a second expression plasmid or may exist as a naked polynucleotide sequence.
  • the plasmid containing such polynucleotide is cut at appropriate restriction enzyme sites ' and cloned into the first expression plasmid which also has been cut at appropriate restriction enzyme sites.
  • the resulting expression plasmid thus includes a polynucleotide which includes the modified retroviral envelope protein.
  • Such plasmid also includes a polynucleotide encoding a minimal signal peptide_of the retroviral envelope protein.
  • minimal signal peptide is meant a signal peptide plus a cleavage site. .
  • polynucleotide as used herein means a polymeric form of nucleotide of any length, and includes ribonucleotides and deoxyribonucleotides. Such term also includes single- and double-stranded DNA, as well as single- and double-stranded RNA. The term also includes modified polynucleotides such as methylated or capped polynucleotides.
  • the retroviral vector particle having a first envelope protein and a modified envelope protein in accordance with the present invention includes a polynucleotide encoding a heterologous polypeptide which is to be expressed in a desired cell.
  • the heterologous polypeptide may, in one embodiment, be a therapeutic agent.
  • therapeutic is used in a generic sense and includes treating agents, prophylactic agents, and replacement agents.
  • polynucleotide encoding the therapeutic agent is under the control of a suitable 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.
  • the polynucleotide encoding the therapeutic agent may be placed into an appropriate retroviral plasmid vector by genetic engineering techniques known to those skilled in the art.
  • the retroviral plasmid vector may be derived from Moloney Murine Leukemia Virus and is of the LN series of vectors, which are described further in Bender, et al., J. Virol., Vol. 61 , pgs. 1639-1649 (1987) and Miller, er a/., Biotechnioues. Vol. 7, pgs 980-990 (1989).
  • Such vectors have a portion of the packaging signal derived from a mouse sarcoma virus, and a mutated gag initiation codon.
  • the term "mutated" as used herein means that the gag initiation codon has been deleted or altered such that the gag protein or fragments or truncations thereof, are not expressed.
  • the retroviral plasmid vector may include at least four cloning, or restriction enzyme recognition sites, wherein at least two of the sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., - 11 -
  • the restriction product has an average DNA size of at least 10,000 base pairs.
  • Preferred cloning sites are selected from the group consisting of Notl, SnaBI, Sail, and Xhol.
  • the retroviral plasmid vector includes each of these cloning sites. Such vectors are further described in U.S. Patent No. 5,672,510, which is incorporated herein by reference in its entirety.
  • a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of Notl, SnaBI, Sail, and Xhol located on the retroviral plasmid vector.
  • the shuttle cloning vector also includes at least one desired polynucleotide encoding a therapeutic agent which is capable of being transferred from the shuttle cloning vector to the retroviral plasmid vector.
  • the shuttle cloning vector may be constructed from a basic "backbone" vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Included in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art.
  • the shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems.
  • the shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria.
  • the shuttle cloning vector may be derived from plasmids such as pBR322; pUC 18; etc.
  • the retroviral plasmid vector includes one or more promoters for the genes contained in the vector.
  • Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ⁇ -actin promoters).
  • CMV human cytomegalovirus
  • viral promoters which may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
  • the polynucleotide encoding the modified retroviral envelope protein is contained in a separate expression vehicle, such as an expression plasmid.
  • the polynucleotide encoding the modified retroviral envelope protein may be - 12 -
  • retroviral plasmid vector for transduction and expression of the modified retroviral envelope protein in producer cell lines.
  • the retroviral plasmid vector which includes a polynucleotide encoding a therapeutic agent, and the expression vehicle including the polynucleotide encoding the modified retroviral envelope protein in accordance with the invention are transduced into a packaging cell line including nucleic acid sequences encoding the gag, pol, and wild-type (i.e., unmodified) env retroviral proteins.
  • packaging cell lines include, but are not limited to, the PE501 , PA317 (ATCC No.
  • the vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, and use of liposomes, such as hereinabove described, and CaPO 4 precipitation.
  • Such producer cells generate infectious retroviral vector particles that include the first, or unmodified wild-type retroviral envelope protein, the modified retroviral envelope protein, and a polynucleotide encoding a therapeutic agent.
  • a packaging cell which includes polynucleotides encoding the gag and pol proteins, a polynucleotide encoding a first retroviral envelope protein free of non-retroviral peptides (which in one embodiment, may be a wild-type retroviral envelope protein), and a polynucleotide encoding the modified retroviral envelope protein.
  • a producer cell for generating retroviral vector particles which include the first and modified envelope proteins in accordance with the present invention is produced by introducing into such packaging cell either a retroviral vector particle or a retroviral plasmid vector, in each case including a polynucleotide encoding a therapeutic agent.
  • the producer cell line thus generates infectious retroviral vector particles including the first retroviral envelope protein and the modified retroviral envelope protein and the polynucleotide encoding the therapeutic agent.
  • the retroviral vector particles which include the first retroviral envelope protein and the modified retroviral envelope protein, and a polynucleotide encoding a therapeutic agent, may be administered to a host in order to express the therapeutic agent in the host.
  • the retroviral vector particles are administered to the host in an amount effective to produce a therapeutic effect in the host.
  • the host may be a mammalian host, which may be a human or non-human primate host.
  • retroviral vector particles are administered to a host for the targeting of desired cells in vivo.
  • the retroviral vector particles upon administration to the host, travel to and transduce the desired target cells, whereby the transduced target cells express the therapeutic agent in vivo.
  • the modified retroviral envelope protein includes a ligand which binds to an antibody
  • the retroviral vector particles upon administration to the host, bind to the antibody through the ligand.
  • the retroviral vector particles and the bound antibody then travel to and transduce target cells which have a receptor which binds to the antibody.
  • retroviral vector particles which may be administered is dependent upon a variety of factors, including the age, sex, and weight of the patient, the target cells which are to be transduced, the therapeutic agent which is to be administered, and the severity of the disorder to be treated.
  • the retroviral vector particles may be administered systemically, such as, for example, by intravenous, intraperitoneal, intracolonic, intratracheal, endotracheal, intranasal, intravascular, intrathecal, intraarterial, intracranial, intramarrow, intravesicular, intrapleural, intradermal, subcutaneous, intramuscular, intraocular, intraosseous, and intrasynovial administration.
  • the retroviral vector particles also may be administered topically.
  • Cells which may be transduced with the retroviral vector particles of the present invention include, but are not limited to, primary cells, such as primary nucleated blood cells, primary tumor cells, endothelial cells, epithelial cells, vascular cells, keratinocytes, stem cells, hepatocytes, chondrocytes, connective tissue cells, fibroblasts and fibroelastic cells of connective tissues, mesenchymal cells, mesothelial cells, and parenchymal cells; smooth muscle cells of the vascuiature; hematopoietic stem cells; T-lymphocytes; B-lymphocytes; neutrophils; macrophages; platelets; erythrocytes; reparative mononuclear granulocytic infiltrates of inflamed tissues; nerve cells; brain cells; muscle cells; osteocytes and osteoblasts in bone; lung cells, pancreatic cells; epithelial and subepithelial cells of the gastrointestinal and respiratory tracts
  • the selection of the particular cells which are to be transduced is dependent upon the disease or disorder to be treated as well as the ligand contained in the second retroviral envelope protein. It is to be understood that the scope of the present invention is not to be limited to the transduction of any specific target cells.
  • Retroviral vector particles of the present invention include, but are not limited to, severe combined immune deficiency caused by adenosine deaminase deficiency; sickle cell anemia; thalassemia; hemophilia A - 14 -
  • diabetes emphysema caused by ⁇ -l-antitrypsin deficiency
  • Alzheimer's disease AIDS
  • chronic granulomatosis Gaucher's disease
  • Lesch-Nyhan syndrome muscular dystrophy, including Duchenne muscular dystrophy
  • Parkinson's disease cystic fibrosis
  • phenylketonuria hypercholesterolemia
  • other illnesses such as growth disorders and heart diseases, such as, for example, those caused by alterations in the way cholesterol is metabolized and defects in the immune system, and other cardiovascular diseases.
  • retroviral vector particles When the modified retroviral envelope protein of the retroviral vector particle includes a ligand which binds to an extracellular matrix component, such retroviral vector particles may be employed in treating diseases or disorders which are associated with an exposed extracellular matrix component. Such diseases or disorders include, but are not limited to, cardiovascular diseases; cirrhosis of the liver; and connective tissue disorders (including those associated with ligaments, tendons, and cartilage), and vascular disorders associated with the exposition of collagen.
  • the retroviral vector particles may be used to deliver therapeutic genes to restore endothelial cell function and to combat thrombosis, in addition to limiting the proliferative and fibrotic responses associated with neointima formation.
  • the retroviral vector particles also may be employed in treating vascular lesions; ulcerative lesions; areas of inflammation; sites of laser injury, such as the eye, for example; sites of surgery; arthritic joints; scars; and keloids.
  • the retroviral vector particles also may be employed in wound healing.
  • retroviral vector particles which include the modified retroviral envelope protein hereinabove described wherein said modified retroviral envelope protein includes a ligand which binds to an extracellular matrix component also may be employed in the treatment of tumors, including malignant and non-malignant tumors.
  • tumors when invading normal tissues or organs, secrete enzymes such as collagenases or metalloproteinases which provide for the exposition of extracellular matrix components.
  • the retroviral vector particles become concentrated at the exposed matrix components which are adjacent the tumor, whereby the retroviral vector particles then infect the tumor cells.
  • Such tumors include, but are not limited to, carcinomas; sarcomas, including chondrosarcoma, osteosarcoma, and fibrosarcoma; and brain tumors.
  • a retroviral vector particle including the modified retroviral envelope protein as hereinabove described and which includes a ligand which binds to an extracellular matrix component located at a tumor site, and a polynucleotide encoding a negative selective marker or "suicide" gene, such as, for - 15 -
  • the Herpes Simplex Virus thymidine kinase (TK) gene may be administered to a patient, whereby the retroviral vector particles transduce the tumor cells.
  • an interaction agent or prodrug such as gancyclovir or acyclovir, is administered to the patient, whereby the transduced tumor cells are killed.
  • the retroviral vector particles which include the first retroviral envelope protein and the modified retroviral envelope protein hereinabove described and a polynucleotide encoding a therapeutic agent, may be administered to an animal in vivo as part of an animal model for the study of the effectiveness of a gene therapy treatment.
  • the retroviral vector particles may be administered in varying doses to different animals of the same species, whereby the retroviral vector particles will transduce the desired target cells in the animal.
  • the animals then are evaluated for the expression of the desired therapeutic agent in vivo in the animal. From the data obtained from such evaluations, one may determine the amount of retroviral vector particles to be administered to a human patient.
  • the retroviral vector particles of the present invention also may be employed in the in vitro transduction of desired target cells, which are contained in a cell culture containing a mixture of cells. Upon transduction of the target cells in vitro, the target cells produce the therapeutic agent or protein in vitro. The therapeutic agent or protein then may be obtained from the cell culture by means known to those skilled in the art.
  • the retroviral vector particles also may be employed for the transduction of cells in vitro in order to study the mechanism of the genetic engineering of cells in vitro.
  • the "escort-protein" which forms the modified retroviral envelope protein may be employed to form proteoliposomes; i.e., the "escort-protein” forms a portion of the liposome wall.
  • proteoliposomes may be employed for gene transfer or for drug delivery to desired target cells.
  • the retroviral vector particles may include, in addition to the first retroviral envelope protein and the modified retroviral envelope protein hereinabove described, one or more additional modified envelope proteins, wherein the non-retroviral protein(s) or peptide(s) which replaces the amino acid residues which were removed from the unmodified envelope protein provides an additional function(s) to the retroviral vector particles.
  • functions include, but are not limited to, complement regulation or - 16 -
  • proteins or peptides which may be placed in the additional retroviral envelope protein(s) include, but are not limited to, complement regulatory proteins or complement resistance proteins such as CD55, CD46, and CD59; immunosuppressive agents such as TGF- ⁇ 1 and lnterleukin-10; and growth factors and cytokines including, but not limited to, EGF, IGF, VEGF, and all interleukins.
  • Such additional modified envelope proteins may be generated by transducing a polynucleotide encoding such a modified envelope protein into a packaging cell as hereinabove described.
  • a retroviral vector particle that may be targeted to a particular cell, and possess additional properties such as those hereinabove described.
  • the retroviral vector particle has, in addition to the first retroviral envelope protein, first and second modified retroviral envelope proteins as hereinabove described.
  • the non-retroviral protein or peptide is a ligand which binds to a desired target molecule.
  • the non-retroviral protein or peptide is a complement regulatory protein.
  • Such a retroviral vector particle may be administered to a host, whereby the retroviral particle is targeted to a desired cell, retains the infectivity of wild-type retrovirus, and is resistant to complement.
  • Synthetic oligonucleotides encoding a collagen binding domain with strategic linkers were generated.
  • the polypeptide including the collagen binding domain and linkers has the following sequence:
  • GHMWREPSFMALSGAS (SEQ ID NO:9).
  • Antisense 3' - GCCGGTATACACCGCGCTTGGCTCGA
  • Antisense 3' - GCCGGTATACACCGCGCTTGGCTCGA AAGTACCGAGACTCGCCACGATCGCGGCC - 5' (SEQ ID NO:13).
  • Antisense 3' - GCCGGTATACACCGCGCTTGGCTCG
  • tandem synthetic oligonucleotides were heated to 95°C and allowed to anneal by gradual cooling to room temperature.
  • the DNA duplexes were separated from single- stranded oligonucleotides by passage through a G25 column (5 Prime ® 3 Prime, Inc., Boulder, Colorado). Agarose gels were used to confirm the purity and conformation of the synthetic oligonucleotide inserts. - 18 -
  • the inserts were cloned into the CEE (ecotropic) - delta hinge env construct (Wu, et al., J. Virol., , July 1998, p 5383-5391 ), which was modified by replacement of an amphotropic hypervariable polyproline or "hinge” region (SEQ ID NO:2) containing three unique restriction sites (Avrll (at codon 1 of the "hinge” region), Pstl (at codon 35 of the "hinge” region), Stul (at codon 48 of the "hinge” region)), and an NgoMI restriction site (at codon 60 of the "hinge” region).
  • SEQ ID NO:2 amphotropic hypervariable polyproline or "hinge” region
  • the vector was cut with the following restriction enzymes to generate the respective constructs: BstEII insert; BstEII to Avrll; BstEII to Pstl; BstEII to Stul; BstEII to NgoMI; and Stul insert.
  • the linearized vectors were confirmed by restriction analysis on agarose gels and purified by the GeneClean method (Bio 101 , Vista, California), prior to ligation with the respective collagen binding domain inserts and T4 DNA ligase (New England Biolabs, Beverly, Massachusetts) for either 3 hours at room temperature or overnight at 4°C.
  • Plasmid DNA was extracted from selected transformed clones using QIA prep Miniprep Kits (Qiagen, Valencia, California). Each construct was confirmed by digestion with the appropriate restriction enzymes described above and analysis of the respective inserts. Restriction analysis was followed by direct DNA sequence analysis using the T7 Sequenase sequencing kit (Amersham Life Science, Inc., Cleveland, Ohio).
  • the plasmids containing the coding sequences for the modified envelope proteins which include the 18 amino acid residues of the N-terminal of the receptor binding region of the ecotropic envelope, the collagen binding domain, a portion of the hypervariable polyproline region of amphotropic envelope protein, the remaining C-terminus of the surface protein, and the transmembrane proteins, sometimes are hereinafter referred to as "pESCORT.”
  • Retroviral vectors bearing "escort" protein constructs were assembled using a four- plasmid transient transfection system modified from Soneoka, et al., Nucleic Acids Research. Vol. 23, pgs. 628-633 (1995), in which the wild-type (amphotropic or ecotropic) envelope was co-expressed.
  • the four plasmids employed were (i) pHIT112; (ii) pHIT60; (iii) one of the pESCORT plasmids; and (iv) either pCAE or pCEE (Morgan, et al., J. Virol., Vol. 67, No. 8, pgs. 4712-4721 (August 1993)).
  • Plasmid pHIT60 provided by Dr. Paula Cannon, University of Oxford, Oxford, United Kingdom, includes the SV40 origin of replication and the retroviral gag-pol gene under the control of a cytomegalovirus (CMV) promoter. Plasmid pHIT112, provided by Ling Li, USC Gene Therapy Laboratories, Los Angeles, California, - 19 -
  • each plasmid includes a LacZ gene under the control of a hybrid CMV-LTR promoter, and a neomycin resistance gene under the control of the SV40 promoter.
  • 10mg of each plasmid were cotransfected by the calcium phosphate method into 293 T cells, which express SV40 large T antigen. (Pear, et al., Proc. Nat. Acad. Sc Vol. 90, pgs. 8392-8396 (September 1993)).
  • the producer cells were treated subsequently with 10mM sodium butyrate for 8 to 12 hours and retroviral supernatants were harvested 24 hours after transfection.
  • the retroviral vector supernatants then were tested (i) for binding affinity to collagen matrices using a modified ELISA (Hall, et al., Human Gene Therapy. Vol. 8, pgs. 2183-2192 (1997)) and (ii) for infectivity by the expression of ⁇ -gaiactosidase activity in NIH 3T3 cells. (Hall, et al., 1997).
  • Viral titers were determined and quantified based on expression of the ⁇ - galactosidase reporter gene. Briefly, 2.5 x 10 4 NIH 3T3 cells were plated in each well of 6 well plates prior to transduction. The medium was replaced with 1 ml of serial dilutions of viral supernatant with 8 mg/ml polybrene for 2 hours. One ml of fresh D10 was added to the cultures, which then were maintained overnight at 37°C and 5% C0 2 . The medium was replaced with fresh D10 and cultures were maintained for an additional 24 hours. Expression of ⁇ -galactosidase in the respective cultures was evaluated by X-gal staining 48 hours after transduction of the NIH 3T3 cells.
  • Figure 1 shows ELISA results for the retroviral vectors WT-CEE, BS-CEE.CEE, BN- CEE.CEE, and BA-CEE.CEE.
  • the vector WT-CEE is a wild-type vector with an ecotropic envelope protein.
  • BS-CEE.CEE is a retroviral vector with a wild-type ecotropic envelope protein, and an "escort protein" envelope protein formed by inserting the collagen binding - 20 -
  • BN- CEE.CEE is a retroviral vector with a wild-type ecotropic envelope protein, and an "escort protein" envelope formed by inserting the collagen binding domain between the BstEII and NgoMI sites of the CEE (ecotropic)-delta hinge env construct.
  • BA-CEE.CEE is a retroviral vector including a wild-type ecotropic envelope protein, and an "escort protein" envelope formed by inserting the collagen binding domain between the Bstll and Avrll sites of the CEE (ecotropic)-delta hinge env construct.
  • the BS-CEE.CEE, BN-CEE.CEE and BA-CEE.CEE vectors bound to the collagen-coated wells.
  • the majority of the receptor binding region of the envelope protein and a portion or all the hypervariable polyproline region could be removed and replaced with a collagen binding domain.
  • the plasmids are co-transfected into 293 T-cells as described in Example 1 , followed by sodium butyrate treatment to produce high titer retroviral vectors.
  • the following retroviral vectors were generated, as described in Table I below:
  • the binding affinity of the Protein A bearing virions for purified IgG was evaluated in comparison to wild type CEE and CAE virions using a modification of standard ELISA techniques described in Hall, 1997, except that the ELISA assay employed the 83A25 rat monoclonal antibody directed against the murine leukemia virus env protein (Evans, et al., J_. Virol.. Vol. 64, No. 12, pgs. 6176-6183(1990)), and the wells were pre-coated with purified human Ig G (Gamma Immune N) instead of collagen Type I.
  • the virions including a wild-type envelope protein, and an "escort protein" in which a portion of the envelope protein is removed and replaced with Protein A, remained bound to IgG (dark staining wells) upon washing with PBS, while the wild-type CEE and CAE virions were removed.
  • Vector PZBA.CAE is identical to PABA.CAE, except that in the "escort protein," protein ZZ (Nilsson, et al., Protein Eng., VOL 1 , pgs. 107-1 13 (1987)), a 1 16 amino acid residue protein which binds to IgG, was inserted between the BstEII and Avrll sites.
  • the ELISA assay was conducted in accordance with the procedure described in Example 2.
  • Wells containing 5 x 10 5 KSY1 Kaposi sarcoma cells were contacted with 1 ,000 ng or 5,000 ng of KDR/Flk-1 antibody, or with Polybrene.
  • One well was contacted with neither material.
  • the cells which were contacted with antibody then were contacted with PABA.CAE or wild-type CAE having a 22
  • the vectors including the "escort proteins” exhibited an antibody-dependent and dose-dependent increase in efficiency of transduction of KDR/Flk-1 antibody-coated endothelial KSY1 Kaposi's sarcoma cells, when compared to vectors including only a wild-type env.
  • the data indicate that transduction efficiency is enhanced by molecular "tethering" of chimeric virions against the endothelial cell surface.
  • IgG-targeted vectors may provide an efficient gene delivery vehicle for delivering genes to endothelial cell receptors in transplanted vascular grafts and organs, including hearts, kidneys, lungs, pancreases, and livers.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biomedical Technology (AREA)
  • Neurosurgery (AREA)
  • Genetics & Genomics (AREA)
  • Neurology (AREA)
  • Virology (AREA)
  • Hematology (AREA)
  • Diabetes (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Plant Pathology (AREA)
  • Psychiatry (AREA)
  • Psychology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Cardiology (AREA)
  • Oncology (AREA)
  • Obesity (AREA)
  • Microbiology (AREA)
  • Communicable Diseases (AREA)

Abstract

L'invention concerne un vecteur rétroviral comportant une première protéine d'enveloppe rétrovirale et au moins une protéine d'enveloppe rétrovirale modifiée. Ladite première protéine d'enveloppe rétrovirale comprend une protéine de surface qui comporte (1) une région de fixation de récepteur; (2) une région de polyproline hypervariable; et (3) une partie de corps. Ladite protéine d'enveloppe rétrovirale modifiée, avant modification, comprend une protéine de surface qui comporte (1) une région de fixation de récepteur; (2) une région de polyproline hypervariable; et (3) une partie de corps. La protéine d'enveloppe rétrovirale modifiée est caractérisée par le fait qu'elle a été modifiée de telle sorte qu'au moins 90 % des résidus d'acides aminés de la région de fixation de récepteur de ladite protéine de surface de ladite protéine d'enveloppe rétrovirale modifiée ont été enlevés et remplacés par une protéine ou un peptide non rétroviral(e).
EP99914700A 1998-04-29 1999-04-28 Vecteurs retroviraux incluant des proteines d'escorte a enveloppe modifiee Ceased EP1073758A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US6939898A 1998-04-29 1998-04-29
US69398 1998-04-29
PCT/IB1999/000764 WO1999055893A1 (fr) 1998-04-29 1999-04-28 Vecteurs retroviraux incluant des proteines d'escorte a enveloppe modifiee

Publications (1)

Publication Number Publication Date
EP1073758A1 true EP1073758A1 (fr) 2001-02-07

Family

ID=22088734

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99914700A Ceased EP1073758A1 (fr) 1998-04-29 1999-04-28 Vecteurs retroviraux incluant des proteines d'escorte a enveloppe modifiee

Country Status (6)

Country Link
EP (1) EP1073758A1 (fr)
JP (1) JP2002514388A (fr)
CA (1) CA2326407C (fr)
IL (1) IL139016A0 (fr)
NZ (2) NZ532894A (fr)
WO (1) WO1999055893A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7078483B2 (en) 1998-04-29 2006-07-18 University Of Southern California Retroviral vectors including modified envelope escort proteins
AU2003230873A1 (en) * 2002-04-11 2003-10-27 University Of Southern California Targeted cytocidal virionoids for antiangiogenesis
EP1769078A1 (fr) * 2004-07-01 2007-04-04 VIRxSYS Corporation Lignee cellulaire d'encapsidation de vecteurs
DK3155004T3 (da) * 2014-06-12 2021-04-06 Univ Do Porto Reitoria Vaccine til immunkompromitterede værter
BR112020014093A2 (pt) * 2018-01-11 2020-12-01 Chameleon Biosciences, Inc. vetores imuno-evasivos e uso para terapia gênica

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU3434393A (en) * 1992-01-17 1993-08-03 Regents Of The University Of Michigan, The Targeted virus
JPH10501403A (ja) * 1994-03-04 1998-02-10 ユニバーシティ オブ メディシン アンド デンティストリー オブ ニュージャージー 抗体−エンベロープ融合タンパク質および野生型エンベロープ融合タンパク質を含むレトロウィルスベクターを用いる細胞型特異的遺伝子移入
US5643770A (en) * 1994-07-21 1997-07-01 Alexion Pharmaceuticals, Inc. Retroviral vector particles expressing complement inhibitor activity
WO1997024446A2 (fr) * 1995-12-29 1997-07-10 Chiron Corporation Ligands de ciblage de vehicules pour l'apport de genes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
OHNO K.; MERUELO D.: "Retrovirus vectors displaying the IgG-binding domain of protein A", BIOCHEMICAL AND MOLECULAR MEDICINE, vol. 62, 1997, pages 123 - 127 *

Also Published As

Publication number Publication date
NZ532894A (en) 2005-10-28
CA2326407C (fr) 2012-09-11
JP2002514388A (ja) 2002-05-21
WO1999055893A1 (fr) 1999-11-04
IL139016A0 (en) 2001-11-25
NZ507645A (en) 2004-06-25
CA2326407A1 (fr) 1999-11-04

Similar Documents

Publication Publication Date Title
US8148509B2 (en) Transgene delivering retrovirus targeting collagen exposed at site of tissue injury
US6117681A (en) Pseudotyped retroviral particles
US6004798A (en) Retroviral envelopes having modified hypervariable polyproline regions
US7078483B2 (en) Retroviral vectors including modified envelope escort proteins
WO1996030504A1 (fr) Polypeptide d'enveloppe virale modifie
CA2326407C (fr) Vecteurs retroviraux incluant des proteines d'escorte a enveloppe modifiee
US6132731A (en) Murine leukemia virus vectors
EP0817860B1 (fr) Particules retrovirales pseudotypees
AU2003200255B2 (en) Retroviral vectors including modified envelope escort proteins
US20060292121A1 (en) Retroviral vectors including modified envelope escort protein
AU3341299A (en) Retroviral vectors including modified envelope escort proteins
AU4386999A (en) Isolated amphiphilic peptides derived from the cytoplasmic tail of viral envelope proteins
US20020058020A1 (en) Isolated amphiphilic peptides derived from the cytoplasmic tail of viral envelope proteins
US7531322B2 (en) Cell-specific retroviral vectors with antibody domains and method for the production thereof for selective gene transfer
Tai Targeting of replication-competent retroviral vectors to HER2-overexpressing breast cancer cells
NZ536851A (en) Vector particles containing TVTM peptides

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20001005

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU NL PT SE

17Q First examination report despatched

Effective date: 20040407

18D Application deemed to be withdrawn

Effective date: 20041019

18RA Request filed for re-establishment of rights before grant

Effective date: 20050606

D18D Application deemed to be withdrawn (deleted)
17Q First examination report despatched

Effective date: 20040407

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED

18R Application refused

Effective date: 20090604