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WO1996030504A1 - Polypeptide d'enveloppe virale modifie - Google Patents

Polypeptide d'enveloppe virale modifie Download PDF

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Publication number
WO1996030504A1
WO1996030504A1 PCT/US1996/003908 US9603908W WO9630504A1 WO 1996030504 A1 WO1996030504 A1 WO 1996030504A1 US 9603908 W US9603908 W US 9603908W WO 9630504 A1 WO9630504 A1 WO 9630504A1
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Prior art keywords
seq
polypeptide
amino acids
receptor
binds
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PCT/US1996/003908
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English (en)
Inventor
W. French Anderson
Yi Zhao
Yawen L. Chiang
Albert J. Mackrell, Jr.
Michael Januszeski
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Genetic Therapy, Inc.
University Of Southern California
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Application filed by Genetic Therapy, Inc., University Of Southern California filed Critical Genetic Therapy, Inc.
Priority to AU53202/96A priority Critical patent/AU5320296A/en
Publication of WO1996030504A1 publication Critical patent/WO1996030504A1/fr

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C07K14/68—Melanocyte-stimulating hormone [MSH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • 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
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    • 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
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
    • C12N2810/6045—RNA rev transcr viruses
    • C12N2810/6054—Retroviridae
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
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    • 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
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    • 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
    • C12N2810/852—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from cytokines; from lymphokines; from interferons
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    • 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
    • C12N2810/854—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from hormones
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    • 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 a polypeptide which is a modified viral envelope.
  • This invention further relates to "targeted" retroviral vector particles. More particularly, this invention relates to retroviral vector particles having a modified, or chimeric, envelope polypeptide wherein a portion of the envelope polypeptide is replaced with a polypeptide which binds to a ligand or receptor of a targeted cell.
  • polypeptide as used herein means a polymer of amino acids and does not refer to any particular length of the polymer. Such term also includes post-translationally modified polypeptides or proteins (e.g., glycosylated, acetylated, phosphorylated, etc.) .
  • Retroviral vector particles are useful agents for introducing polynucleotides into cells, such as eukaryotic cells.
  • introducing encompasses a variety of methods of transferring polynucleotides into a cell, such methods including transformation, transduction, transfection, and transinfection.
  • Retroviruses typically have three common open reading frames, gag, pol, and env, which encode the structural proteins, encode enzymes including reverse transcriptase, and encode envelope proteins, respectively.
  • retroviral vector particles are produced by packaging cell lines that provide the necessary gag, pol, and env gene products in trans . (Miller, et al. , Human Gene Therapy. Vol. 1, pgs. 5-14 (1990)) . This approach results in the production of retroviral vector particles which transduce mammalian cells, but are incapable of further replication a ter they have integrated into the genome of the cell.
  • 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 cells are returned to the patient with the engineered cells 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.
  • env encoded by the env gene
  • purification necessitates undesirable manipulations of the cells and may be problematic in situations in which the preferred target cells either are difficult to purify or are present at low or variable frequencies in mixed cell populations.
  • retroviral vector particles which could infect particular types of mammalian cells.
  • Figure 1 is a schematic of the polypeptide (SEQ ID NO:l) ;
  • Figure 2 is a schematic of the polypeptide (SEQ ID NO:2) ;
  • Figure 3 is a graph of relative percentage of melanin produced by B16-F1 cells stimulated with media from C0S7 cells transfected with one of plasmids pcDNA-EF, p3-l, p3-2, or p6-3.
  • retroviral vector particles can be targeted to desired cells by providing the retroviral vector particle with a chimeric polypeptide which is derived from a viral envelope. More particularly, the chimeric polypeptide is produced by deleting specific portions of the polypeptide which comprises the receptor binding portion of a viral envelope hereinafter described, and replacing the deleted portions, hereinafter described, with a targeting polypeptide which binds to a receptor or ligand on the targeted cells.
  • a retroviral vector particle having a modified envelope polypeptide for targeting the retroviral vector particle to cells.
  • the envelope Prior to modification, includes a receptor binding region which is a polypeptide selected from the group consisting of
  • SEQ ID NO:3 which is the receptor binding region of 10A1 murine leukemia virus envelope
  • SEQ ID NO:4 which is the receptor-binding region of murine leukemia virus NZB-9-1 xenotropic envelope
  • SEQ ID NO:5 which is the receptor-binding region of murine leukemia virus polytropic MX27 provirus envelope.
  • the ecotropic envelope includes the polypeptide having (SEQ ID N0:1), in the modified envelope at least a portion of (i) amino acids 70 to 92 of (SEQ ID NO:l) ; or (ii) amino acids 44 to 114 of (SEQ ID N0:1) ; or (iii) amino acids 44 to 131 of (SEQ ID NO:l); or (iv) amino acids 17 to 182 of (SEQ ID N0:1) are replaced with a targeting polypeptide which binds to a ligand or receptor on the targeted cells.
  • the amphotropic envelope includes the polypeptide having (SEQ ID NO:2)
  • the modified envelope at least a portion of (i) amino acids 47 to 75 of (SEQ ID NO:2) ; or (ii) amino acids 47 to 93 of (SEQ ID NO:2) ; or (iii) amino acids 47 to 163 of (SEQ ID NO:2) are replaced with a targeting polypeptide which binds to a ligand or receptor on the targeted cells.
  • the envelope When, prior to modification, the envelope includes the polypeptide having (SEQ ID NO:3) , in the modified envelope at least a portion of (i) amino acids 47 to 75 of (SEQ ID NO:3) ; or (ii) amino acids 47 to 93 of (SEQ ID NO:3); or (iii) amino acids 47 to 163 of (SEQ ID NO:3) are replaced with a targeting polypeptide which binds to a ligand or receptor on the targeted cells.
  • the xenotropic envelope includes the polypeptide having (SEQ ID NO:4)
  • the modified envelope at least a portion of (i) amino acids 47 to 74 of (SEQ ID NO:4) ; or (ii) amino acids 47 to 92 of (SEQ ID NO:4) ; or (iii) amino acids 47 to 154 of (SEQ ID N0:4) are replaced with a targeting polypeptide which binds to a ligand or receptor on the targeted cells.
  • the polytropic envelope includes the polypeptide having (SEQ ID NO:5) , in the modified envelope at least a portion of (i) amino acids 47 to 70 of (SEQ ID NO:5) ; or (ii) amino acids 47 to 88 of (SEQ ID NO:5) ; or (iii) amino acids 47 to 151 of (SEQ ID NO:5) are replaced with a targeting polypeptide which binds to a ligand or receptor on the targeted cells.
  • the envelope prior to modification, includes the polypeptide having (SEQ ID NO:l) , and at least a portion, of amino acids 70 to 92 of (SEQ ID NO:l) are replaced with a targeting polypeptide which binds to a ligand or receptor on the targeted cells.
  • all of amino acids 70 to 92 of (SEQ ID N0:1) are replaced with a targeting polypeptide.
  • at least a portion of amino acids 74 to 91 of (SEQ ID NO:l) are replaced with a targeting polypeptide.
  • all of amino acids 74 to 91 of (SEQ ID N0:1) are replaced with a targeting polypeptide.
  • amino acids 80 to 88 of (SEQ ID NO:l) are replaced with a targeting polypeptide.
  • amino acids 82 to 84 of (SEQ ID NO:l) are replaced with a targeting polypeptide.
  • amino acids 74 to 80 (of SEQ ID NO:l) are replaced with a targeting polypeptide.
  • at least a portion, and preferably all, of amino acids 44 to 114 of (SEQ ID NO:l) are replaced with a targeting polypeptide.
  • at least a portion, and preferably all, of amino acids 44 to 131 of (SEQ ID NO:l) are replaced with a targeting polypeptide.
  • at least a portion, and preferably all, of amino acids 17 to 182 of (SEQ ID NO:l) are replaced with a targeting polypeptide.
  • the envelope prior to modi ication, includes the polypeptide having (SEQ ID NO:2) , and at least a portion, and preferably all, of amino acids 47 to 75 of (SEQ ID NO:2) is replaced with a targeting polypeptide which binds to a ligand or receptor on the targeted cells.
  • at least a portion, and preferably all, of amino acids 47 to 93 of (SEQ ID NO:2) are replaced.
  • at least a portion, and preferably all, of amino acid residues 47 to 163 of (SEQ ID NO:2) are replaced.
  • the envelope prior to modification, includes the polypeptide having (SEQ ID NO:3) , and at least a portion, and preferably all of amino acids 47 to 75 of (SEQ ID NO:3) is replaced with a targeting polypeptide which binds to a ligand or receptor on the targeted cells.
  • at least a portion, and preferably all, of amino acids 47 to 93 of (SEQ ID NO:3) is replaced.
  • at least a portion, and preferably all, of amino acids 47 to 163 of (SEQ ID NO:3) is replaced.
  • the envelope prior to modification, includes the polypeptide having (SEQ ID NO:4) , and at least a portion, and preferably all, of amino acids 47 to 74 of (SEQ ID NO:4) is replaced with a targeting polypeptide which binds to a ligand or receptor on the targeted cells.
  • at least a portion, and preferably all, of amino acids 47 to 92 of (SEQ ID NO:4) is replaced.
  • at least a portion, and preferably all, of amino acids 47 to 154 of (SEQ ID NO:4) is replaced.
  • the envelope prior to modification, includes the polypeptide having (SEQ ID NO:5) , and at least a portion, and preferably all, of amino acids 47 to 70 of (SEQ ID NO:5) is replaced with a targeting polypeptide which binds to a ligand or receptor on the targeted cells.
  • at least a portion, and preferably all, of amino acids 47 to 88 of (SEQ ID NO:5) is replaced.
  • at least a portion, and preferably all, of amino acids 47 to 151 of (SEQ ID NO:5) is replaced.
  • polypeptides (SEQ ID NO:l) through (SEQ ID NO:5) are portions of a protein known as gp 70, which is included in the envelope of murine leukemia viruses.
  • gp 70 protein includes the following regions: (i) the secretory signal or "leader" sequence; (ii) the receptor binding region; (iii) the hinge region; and (iv) the body portion.
  • the polypeptides (SEQ ID NO:l) and (SEQ ID NO:2) are receptor binding regions of ecotropic envelope of Moloney Murine Leukemia Virus; and amphotropic retroviral envelope of 4070A retrovirus, respectively.
  • SEQ ID NO:3 is the receptor binding region of the 10A1 murine leukemia virus envelope, and also is described in Ott, et al. , J. Virol.. Vol. 64, pgs. 757-766 (1990) .
  • the nucleic acid sequence of such envelope is registered as GenBank Accession No. M33470.
  • SEQ ID NO:4 is the receptor binding region of murine leukemia virus NZB-9-1 xenotropic envelope, the nucleic acid sequence of which is registered as GenBank Accession No. KO2730 and described in O'Neill, et al. , J. Virol.. Vol. 53, pgs. 100-106 (1985) .
  • SEQ ID NO:5 is the receptor binding region of mouse murine leukemia virus polytropic MX27 provirus, the nucleic acid sequence of which is registered as GenBank Accession No. M17326, and is described in Stoye, et al., J. Virol.. Vol. 61, pgs. 2659-2669 (1987) .
  • retroviruses can be made "targetable" to a specific type of cell if a portion of the receptor binding region is modified such that the receptor binding region includes a polypeptide which binds to a ligand or receptor of a target cell.
  • retroviral vector particles of the present invention include a modified receptor binding region of the envelope protein
  • retroviral particles also may have additional modifications in other regions of the envelope protein, such as, for example, the secretory signal or "leader" sequence, the hinge region, or the body portion.
  • modifications may include deletions and/or substitutions.
  • Targeting polypeptides which may be employed include, but are not limited to, antibodies and fragments thereof, including single-chain antibodies, monoclonal antibodies, and polyclonal 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 polymorphic epithelial mucin found on ovarian cancer cells; antibodies to / S-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 anti
  • cytokines include, but are not limited to, interleukins, including Interleukin-l ⁇ , Interleukin 1/3, and Interleukins 2 through 14; growth factors such as epithelial growth factor (EGF) , TGF-CK, TGF- / S, 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-a, interferon-3, and interferon- ⁇ ; inhibin A; inhibin B; chemotactic factors; ⁇ -type intercrine cytokines; and S-
  • EGF epithelial growth factor
  • TGF-CK
  • Still other targeting polypeptides which may be employed include, but are not limited to, melanotropin stimulating hormones, which bind to the MSH receptor on melanoma cells, such as, for example, alpha-melanotropin stimulating hormone or alpha-MSH; erythropoietin, which binds to the erythropoietin receptor; adhesins; selectins; CD34, which binds to the CD34 receptor of hematopoietic stem cells; CD33, which binds to premyeloblastic leukemia cells,- stem cell factor; integrins; asialoglycoproteins, including asialoorosomucoid, asialofetuin, and alpha-1 acid glycoprotein, which binds to the asialoglycoprotein receptor of liver cells; insulin; glucagon; gastrin polypeptides, which bind to receptors on hematopoietic stem cells; C-kit ligand; tumor necrosis factors (or
  • the envelope includes a polypeptide selected from the group consisting of (a) a polypeptide having the sequence (SEQ ID NO:l) ; (b) a polypeptide having the sequence (SEQ ID NO:2) ; (c) a polypeptide having the sequence (SEQ ID NO:3) ; (d) a polypeptide having the sequence (SEQ ID N0:4) ; and (e) a polypeptide having the sequence (SEQ ID NO: 5) .
  • the ecotropic envelope includes (SEQ ID NO:l) , in the modified polynucleotide, at least a portion of (i) the polynucleotide encoding amino acids 70 to 92 of (SEQ ID NO:l) ; or (ii) the polyncleotide encoding amino acids 47 to 93 of (SEQ ID NO:3) ; or (iii) the polynucleotide encoding amino acids 44 to 114 of (SEQ ID NO:l) ; or (iii) the polynucleotide encoding amino acids 44 to 131 of (SEQ ID NO:l) ; or (iv) the polynucleotide encoding amino acids 17 to 182 of (SEQ ID NO:l) is removed and replaced with a polynucleotide encoding a targeting polypeptide which binds to a ligand or receptor on the targeted cells.
  • the amphotropic envelope includes (SEQ ID NO:2) , in the modified polynucleotide at least a portion of (i) the polynucleotide encoding amino acids 47 to 75 of (SEQ ID NO:2); or (ii) the polynucleotide encoding amino acids 47 to 93 of (SEQ ID NO:2) ; or (iii) the polynucleotide encoding amino acids 47 to 163 of (SEQ ID NO:2) is replaced with a polynucleotide encoding a targeting polypeptide which binds to a ligand or receptor on the targeted cells.
  • the envelope includes the polypeptide having (SEQ ID NO:3) , in the modified polynucleotide at least a portion of (i) the polynucleotide encoding amino acids 47 to 75 of (SEQ ID NO:3) ; or (ii) the polynucleotide encoding amino acids 47 to 93 of (SEQ ID NO:3) ; or (iii) the polynucleotide encoding amino acids 47 to 163 of (SEQ ID NO:3) is removed and replaced with a polynucleotide encoding a targeting polypeptide which binds to a ligand or receptor on the targeted cells.
  • the xenotropic envelope includes the polypeptide having (SEQ ID NO:4) , in the modified polynucleotide at least a portion of the polynucleotide encoding (i) amino acids 47 to 74 of (SEQ ID NO:4) ; or (ii) the polynucleotide encoding amino acids 47 to 92 of (SEQ ID NO: ) ; or (iii) the polynucleotide encoding amino acids 47 to 154 of (SEQ ID NO:4)is removed and replaced with a polynucleotide encoding a targeting polypeptide which binds to a ligand or receptor on the targeted cells.
  • the polytropic envelope includes the polypeptide having (SEQ ID NO:5) in the modified polynucleotide at least a portion of (i) the polynucleotide encoding amino acids 47 to 70 of (SEQ ID NO:5) ; or (ii) the polynucleotide encoding amino acids 47 to 88 of (SEQ ID NO:5) ; or. (iii) the polynucleotide encoding amino acids 47 to 151 of (SEQ ID NO:5) is removed and replaced with a polynucleotide encoding a targeting polypeptide which binds to a ligand or receptor on the targeted cells.
  • the polypeptide (SEQ ID NO:l) is encoded by the polynucleotide having (SEQ ID NO:6) or a derivative or analogic thereof.
  • the polypeptide (SEQ ID NO:2) is encoded by the polynucleotide having (SEQ ID NO:7) or a derivative or analogue thereof.
  • the polypeptide (SEQ ID NO:3) is encoded by the polynucleotide having (SEQ ID NO: 8) or a derivative or analogue thereof.
  • the polypeptide (SEQ ID NO:4) is encoded by the polynucleotide having (SEQ ID NO:9) or a derivative or analogue thereof.
  • the polypeptide (SEQ ID NO:5) is encoded by the polynucleotide having (SEQ ID NO:10) or a derivative or analogue thereof.
  • derivative or analogue thereof as used herein means that the polynucleotides encoding one of the polypeptides (SEQ ID NO:l) through (SEQ ID NO:5) may have a sequence different from one of polynucleotides (SEQ ID NO:6) through (SEQ ID NO:10) , yet encode the same polypeptide. Such differences in the polynucleotide sequences may, for example, be due to the degeneration of the genetic code.
  • Such a polynucleotide may be constructed by genetic engineering techniques known to those skilled in the art.
  • a first expression plasmid may be constructed which includes a polynucleotide encoding the unmodified envelope.
  • the plasmid then is engineered such that a polynucleotide encoding an amino acid sequence as hereinabove described may be removed, and to provide appropriate restriction enzyme sites for removal of the polynucleotide sequence encoding an amino acid sequence as hereinabove described, and replacement of such polynucleotide sequence with a polynucleotide sequence encoding a targeting polypeptide.
  • the polynucleotide encoding the targeting polypeptide may be contained in a second expression plasmid or may exist as a naked polynucleotide sequence.
  • the polynucleotide encoding the targeting polypeptide or 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 encoding the modified envelope protein.
  • Such polynucleotide then may be cloned out of the expression plasmid, and into a retroviral plasmid vector.
  • the resulting retroviral plasmid vector which includes the polynucleotide encoding the modified envelope protein, and which also may include a polynucleotide encoding a heterologous protein or peptide, is transfected into an appropriate packaging cell line to form a producer cell line for generating retroviral vector particles including the modified envelope protein.
  • a naked polynucleotide sequence encoding the modified envelope protein is transfected into a "pre ⁇ packaging" cell line including nucleic acid sequences encoding the gag and pol proteins, thereby forming a packaging cell line, or is transfected into a packaging cell line including nucleic acid sequences encoding the gag, pol, and wild-type (i.e., unmodified) env proteins, thereby forming a packaging cell line including nucleic acid sequences encoding wild-type env protein and the modified envelope protein.
  • Such packaging cells then may be transfected with a retroviral plasmid vector, which may include a nucleic acid sequence encoding a heterologous protein or peptide, thereby forming a producer cell line for generating retroviral vector particles including the modified envelope protein.
  • a polynucleotide thus may be contained in the above-mentioned retroviral vector particle, or in a producer cell for generating the above-mentioned retroviral vector particle.
  • 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 modified envelope in accordance with the invention includes a polynucleotide encoding a heterologous polypeptide which is to be expressed in a targeted 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.
  • Polynucleotides encoding therapeutic agents which may be contained in the retroviral vector particle include, but are not limited to, polynucleotides encoding tumor necrosis factor (TNF) genes, such as TNF- ⁇ ; genes encoding interferons such as Interferon-c-, Interferon- ⁇ , and Interferon- ⁇ ; genes encoding interleukins such as IL-1, IL-13, and Interleukins 2 through 14; genes encoding GM-CSF; genes encoding adenosine deaminase, or ADA; genes which encode cellular growth factors, such as lymphokines, which are growth factors for lymphocytes; genes encoding epidermal growth factor (EGF) , and keratinocyte growth factor (KGF) ,- genes encoding soluble CD4; Factor VIII; Factor IX; cytochrome b; glucocerebrosidase; T-cell receptors; the LDL receptor, ApoE, ApoC
  • the polynucleotide encoding the therapeutic agent is under the control of a suitable promoter.
  • suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; the cytomegalovirus (CMV) promoter; the Rous Sarcoma Virus (RSV) promoter; the histone promoter; the polIII promoter, the J-actin promoter; inducible promoters, such as the MMTV promoter, the metallothionein promoter; heat shock promoters; adenovirus promoters; the albumin promoter; the ApoAI promoter; B19 parvovirus promoters; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; human growth hormone promoters, and the MxIFN inducible promoter.
  • the promoter also may be the native promoter which controls
  • polynucleotides encoding the modified envelope polypeptide and 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, such as those hereinabove mentioned, and described further in Bender, et al . , J. Virol. , Vol. 61, pgs. 1639-1649 (1987) and Miller, et al . , Biotechni ⁇ ues. 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., 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 NotI, SnaBI, Sail, and Xhol.
  • the retroviral plasmid vector includes each of these cloning sites. Such vectors are further described in U.S. Patent Application Serial No. 08/340,805, filed November 17, 1994, and in PCT Application No. W091/10728, published July 25, 1991, and incorporated herein by reference in their entireties.
  • 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 NotI, 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. 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 . , Biotechni ⁇ ues. 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 /8-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 retroviral plasmid vector which includes a polynucleotide encoding the modified envelope and a polynucleotide encoding a therapeutic agent, is employed to transduce a packaging cell line to form a producer cell line, which will generate infectious retroviral vector particles.
  • the packaging cell line is a "pre ⁇ packaging" cell line which includes polynucleotides encoding the gag and pol retroviral proteins, but not the envelope, or env, protein. Examples of such "pre-packaging" cell lines include, but are not limited to, GP8 cells, GPL cells, and GPNZ cells as described in Morgan, et al . , ______________ , Vol.
  • Such cell lines upon transduc ion with the retroviral plasmid vector, generates infectious retroviral particles including the modified, or chimeric, envelope and a polynucleotide encoding the therapeutic agent.
  • a retroviral plasmid vector which includes a polynucleotide encoding a modified polynucleotide encoding a modified envelope polypeptide in accordance with the invention and a polynucleotide encoding a therapeutic agent is used to transduce 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 CaP0 4 precipitation.
  • Such producer cells generate infectious retroviral vector particles which include the modified envelope, the wild-type retroviral envelope, a polynucleotide encoding the modified, or chimeric, envelope, and a polynucleotide encoding a therapeutic agent.
  • a packaging cell which includes a nucleic acid sequence encoding a modified chimeric envelope in accordance with the invention, and which may further include nucleic acid sequences encoding the gag and pol proteins.
  • a producer cell for generating viral particles which includes a modified envelope in accordance with the 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 particles including the modified chimeric envelope and the polynucleotide encoding the therapeutic agent .
  • the retroviral vector particles which include the modified envelope, and a polynucleotide encoding a therapeutic agent, may be administered to a 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.
  • 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 exact dosage of 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 sytemically, such as, for example, by intravenous, or intraperitoneal administration, as well as by intranasal, intratracheal, endotracheal, intraarterial, intravesicular, or intracolonic administration.
  • 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, keratinocytes, stem cells, hepatocytes, connective tissue cells, fibroblasts, mesenchymal cells, mesothelial cells, and parenchymal cells; stem cells, such as hematopoietic stem cells; T-lymphocytes; B-lymphocytes; neutrophils; macrophages; platelets; erythrocytes; nerve cells; brain cells; muscle cells; lung cells, pancreatic cells; and malignant and non-malignant tumor cells.
  • primary cells such as primary nucleated blood cells, primary tumor cells, endothelial cells, epithelial cells, keratinocytes, stem cells, hepatocytes, connective tissue cells, fibroblasts, mesenchymal cells, mesothelial cells, and paren
  • 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 targeting polypeptide contained in the modified envelope. 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.
  • Diseases or disorders which may be treated with the 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; 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; and 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.
  • the retroviral vector particles also may be employed in the treatment of tumors, including malignant and non- malignant tumors.
  • a retroviral vector particle including a modified envelope protein, including a targeting polypeptide which binds to a tumor cell, and a polynucleotide encoding a negative selective marker or "suicide" gene, such as, for example, the Herpes Simplex thymidine kinase (TK) gene may be administered to a patient, whereby the retroviral vector particles transduce the tumor cells.
  • an interaction agent such as gancyclovir or acyclovir, is administered to the patient, whereby the transduced tumor cells are killed.
  • the retroviral vector particles which include the modified envelope protein 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 vi tro transduction of desired target cells, which are contained in a cell culture containing a mixture of cells. Upon transduction of the target cells in vi tro, the target cells produce the therapeutic agent or protein in vi tro. 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 vi tro in order to study the mechanism of the genetic engineering of cells in vi tro .
  • modified envelope polypeptides of the present invention may be employed to form proteoliposomes; i.e., the modified envelope polypeptide forms a portion of the liposome wall.
  • proteoliposomes may be employed for gene transfer or for drug delivery to desired target cells.
  • AD422 GTCAAAAGTACGCGTCCGCTGTCTCC mutagenic oligo to create Mlul site in CAE
  • AD880 GGCCGGCTGGGCCCCTCCCCCCGGGGGAGA same as AD879 AGAAAAAGG
  • AD881 ACCTCCCTCGCGGCCGCCACCCCTCGGTGC oligo for SOE PCR to create AACACTGC NotI in E/S2N1
  • AD882 CCGAGGGGTGGCGGCCGCGAGGGAGGTTAA same as AD881 AGGTTCTTCG
  • AD884 ATAAAATCCGGCGGCCGCCTCATTTGATTTA same as AD883 TGAGTTGTCTGG
  • AD890 CGCGTTCCTCCACTGGCGGCCGCA bottom strand complement of AD889
  • AD936 GTTACCCCGGCCGGCTGGGCCCCG bottom strand complement of AD937
  • Cee+ is a CMV-env expression vector constructed by digesting CEE (Morgan, et al . , 1993) with Hind III and Not I, filling in the Not I site with Klenow fragment, and ligating the CMV-env cassette into Bluescript II SK+ digested with Sma I and Hind III.
  • Cee+ ⁇ Not was made by digesting Cee-i- with Not I, removing the 5' -extensions by digesting with Mung Bean Nuclease and recircularizing the plasmid.
  • Single stranded phagemid DNA was made from E. coli JM109 transformed with Cee+ ⁇ Not. 5 ⁇ g of single-stranded DNA was mixed with 4pmol each of AD191 and AD196, and used as template for site directed mutagenesis to introduce Bgl II and Mun I sites.
  • a plasmid, CEC, that had obtained the desired Bgl II and Mun I sites was identified by digestion with these enzymes, and confirmed by DNA sequencing. This plasmid served as the basis of all chimeric envelope expression vectors.
  • CEC was digested with Xma I and dephosphorylated.
  • a phosphorylated double-stranded linker made by heating and annealing AD249 and AD250, was ligated into this site.
  • the resulting product was then digested with Not I, diluted and recircularized by ligation.
  • CECX a plasmid containing a single copy of the polylinker oriented so that the Sfi I site was closer to the 5'-end of env, was identified by digestion with Sfi I and Not I and confirmed by DNA sequencing.
  • linker was added to Bluescript II SK(+) that had been digested with Xma I and dephosphorylated. Following ligation of the linker, the ligation mix was digested with Not I, ethanol precipitated, diluted and religated. pBSR(L) (for "linker”) was identified by screening for the presence of an Sfi I site and the absence of an Spe I site, and confirmed by sequencing. Addition of linker sequences to e23 scFv.
  • DNA encoding e23 scFv single chain antibody which is an Erb-B2 single chain monoclonal antibody which is to be employed in producing the chimeric envelope polypeptide for targeting a retroviral vector to cells containing an Erb-B2 receptor, and is described in Batra, et al. , Proc. Nat. Acad. Sci.. Vol. 89, pgs. 5867-5871 (1992) , was amplified by mixing 125ng of such DNA and 60pmoles each of AD275 and AD276 in a standard lOO ⁇ l PCR reaction. The product was ethanol precipitated, resuspended and digested with Sfi I and Not I .
  • the digest was electrophoresed through 1.0% Agarose and the digested product was excised from the gel and recovered by electroelution and ethanol precipitation. This fragment was ligated to pBSR(L) that had been digested with Sfi I and Not
  • pBSR-e23 was isolated by screening for the presence of insert by digestion. It was sequenced in its entirety to ensure it was free of PCR introduced mutations.
  • Single-strand pCAE (Morgan, et al . , 1993) was used as a template for site-directed mutagenesis using AD423 to remove the EcoRI site in pCAE (at bp 2,105) to form pCAE/RI .
  • pCAE/RI was linearized by Not I, and then filled in with Klenow fragment, and the plasmid was recircularized, thus forming pCAE/2#.
  • Single-stranded pCAE was used as a template, and site- directed mutagenesis was employed using oligonucleotides AD421 and AD422 to create AvrII (at bp 122) and Mlul (at bp 354) sites, thereby forming pCAE/AM.
  • Single-stranded pCEE+ was used as a template, and oligonucleotide AD604 was used in site-directed mutagenesis to create a Bgl II site in pCee+ (at bp 1385) to form pCee+/BglII .
  • oligonucleotide AD605 was used in site-directed mutagenesis to create an EcoNI site in pCee+ (at bp 1568) to form pCee+/EcoNI.
  • pCee+/BglII and pCee+/EcoNI both were digested with BstXI, and the 2,021 bp fragment from pCee+/EcoNI was ligated to the 4,333 bp fragment from pCee+/BglII to form pCEE+/BN.
  • pCee+/BN then was digested with Not I, filled in, and recircularized to form pCee+/BN ⁇ N' .
  • Both pCee+ and pCee+/BN ⁇ N' were digested by BstEII and BspEI, and the 586 bp fragment from pCee+/BN ⁇ N' was ligated to the 5,768 bp fragment from pCee-f- to form the backbone plasmid pCee+/BN ⁇ N.
  • the plasmid was identified by restriction enzyme digestion and confirmed by DNA sequencing. III. Introduction of Sfil and Not I sites into envelope sequence.
  • oligonucleotides AD870, AD871, AD422, and AD416 were used to introduce an Sfil site at the 5' -end of pCAE/3# (at bp 266) .
  • the PCR amplified fragment was digested by Avrll and Mlul and cloned into pCAE/3# digested with corresponding enzymes to form pA/Sl.
  • the plasmid was identified by Sfi I digestion and DNA sequencing.
  • pA/Sl was linearized by digestion with Mlul and dephosphorylated.
  • a phosphorylated double-stranded linker, made by heating and annealing AD889 and AD890 was ligated into this site to form A/S1N1.
  • the clones were screened by PCR and sequenced.
  • oligonucleotides AD887 , AD888, AD109 and AD111 were used to introduce NotI site at the 3'-end of A/SI (corresponding to bp407 of CAE) .
  • the PCR amplified fragment was digested by Avrll and Mlul and cloned into A/SI digested with the corresponding enzymes to form A/S1N2. Plasmid was identified by Sfil and Not I digestion and DNA sequencing.
  • A/SI was linearized by digestion with EcoRI and dephosphorylated.
  • a phosphorylated double stranded linker, made by heating and annealing AD938 and AD939 was ligated into this site to form A/S1N3.
  • Resultant clones were screened by PCR and sequenced.
  • Cee+/BN ⁇ N was linearized by digestion with BstEII and dephosphorylated.
  • a phosphorylated double stranded linker, made by heating and annealing AD936 and AD937 was ligated into this site to introduce an Sfil site at 1316bp of Cee+ to form E/SO.
  • Resultant clones were screened by PCR and sequenced.
  • Cee+/BN ⁇ N was linearized by digestion with Bglll and dephosphorylated.
  • oligos AD879, AD880, AD725 and AD287 were used to introduce an Sfil site at the 5'-end of Cee+/BN ⁇ N (1475bp) .
  • the PCR amplified fragment was digested by BglII and EcoNI and cloned into Cee+/BN ⁇ N digested with the corresponding enzyme to form E/S2. Plasmid was identified by Sfil digestion and DNA sequencing.
  • oligos AD881, AD882, AD15 and AD16 were used to introduce a NotI site at the 3 '-end of E/S2 (1544bp) .
  • the PCR amplified fragment was digested by Sfil and BspEI and cloned into E/S2 digested with the corresponding enzyme to form E/S2N1. Plasmid was identified by Sfil and NotI digestion and DNA sequencing.
  • oligos AD883, AD884, AD15 and AD16 were used to introduce a NotI site at the 3 '-end of E/Sl (1610bp) .
  • the PCR amplified fragment was digested by Sfil and BspEI and cloned into E/Sl digested with the corresponding enzyme to form E/S1N2. Plasmid was identified by Sfil and NotI digestion and DNA sequencing.
  • oligos AD885, AD886, AD15 and AD16 were used to introduce a NotI site at the 3' -end of E/Sl (1661bp) .
  • the PCR amplified fragment was digested by Sfil and BspEI and cloned into E/Sl digested with the corresponding enzyme to form E/S1N3. Plasmid was identified by Sfil and NotI digestion and DNA sequence. IV. Construction of e23FV-env Chimeras into different expression vectors
  • Chimeric envelope proteins were constructed by replacing a discrete segment of the envelope gene with the sequences encoding the e23Fv.
  • the plasmid pBSR-e23 was digested with Sfi I and Not I, and a fragment containing the e23Fv sequences was isolated. Plasmids A/S1N1, A/S1N2, A/S1N3, E/S2N1, E/N1S2, and E/S1N3 were digested with Sfi I and Not I, and each digested plasmid was separately ligated to an aliquot of the e23Fv fragment to yield the chimeras named ChAl, ChA2, ChA3 , ChEl, ChE2, and ChE3 , respectively.
  • B. Construction of e23FV-env chimeras into LEESN (Ecotropic envelope protein was cloned into LXSN vector)
  • the plasmid LEESN was obtained from Jack Ragheb (NIH) .
  • This plasmid was constructed by digesting CEE (Morgan et al. , 1993) with EcoRI and isolating the fragment encoding the Moloney Murine Leukemia Virus envelope protein. This fragment was ligated to LXSN (Genbank Accession #M28248) that had been digested with EcoRI.
  • This plasmid was digested with Clal and Sail to remove sequences extending from within the env cytoplasmic domain through the end of the 3' -untranslated sequences.
  • the chimeras ChEl, ChE2 and ChE3 were each digested with EcoRI and Clal and the fragment encoding e23-env was isolated.
  • LEESN was digested with EcoRI and Clal and the fragment encoding the LTRs and other vector sequences was ligated to the fragment derived from each of the chimeras (ChEl, ChE2, ChE3) to yield LChElSN, LChE2SN, and LChE3SN.
  • the CAE-derived chimeras (ChAl, ChA2, ChA3) were each digested with Xbal and incubated with Klenow to fill in the 5' -extension.
  • LEESN was digested with EcoRI, incubated with Klenow, and subsequently digested with Clal.
  • the fragment containing the retroviral vector sequences of LEESN was ligated to the isolated fragments from ChAl, ChA2, and ChA3 to yield respectively LChAlSN, LChA2SN, and LChA3SN.
  • Psi-2 cells obtained from ATCC were grown in D10 medium (Dulbecco's Modified Eagles' Medium with 4,500 g/1 glucose and sodium pyruvate, supplemented with 10% FCS and 2 mM glutamine) .
  • the cells were plated at a concentration of 6 x 10 5 cells per 100 mm plate, and cultured for 16-24 hours. The medium was replaced 4 hours prior to transfection.
  • the cells were transfected with LChAlSN, LChA2SN, LChA3SN, LChElSN, LChE2SN, or LChE3SN.
  • Calcium phosphate precipitate was made using a DNA Transfection Kit (5' ⁇ 3'; Boulder, Colorado) .
  • D10 containing precipitate was replaced for 8 hours, at which time the medium was changed to the appropriate selective media containing D10 and G418 at 0.8 mg/ml. Selection with G418 continued for 8-10 days.
  • the medium (containing D10 and G418) was pipetted from transfected cell monolayers of IV.C. above and filtered through 0.45 ⁇ m syringe filters (Millipore) and stored at - 70°C. In order to concentrate the collected retroviral supernatant, the supernatant was spun at 4°C in centriprep 100 ultra-filtration units (Amicon) at 450 xg until the desired volume was attained.
  • T47D cells obtained through transfection of LChAlSN, LChA2SN, LChA3SN, LChElSN, LChE2SN, and LChE3SN (IV.C. above) are subjected to an FACS binding assay to measure e23-mediated binding to T47D or SK-BR-3 cells.
  • T47D cells obtained from ATCC
  • Sk-BR-3 cells obtained from ATCC
  • T47D cells were grown in D10 and F12 (supplemented with 10% FCS and 2mM glutamine) in the ratio of 1:1.
  • SK-BR-3 cells were grown in MclO (McCoy's 5A medium supplemented with 10% FCS and 2 mM glutamine) . e23-mediated binding to cells was measured using a derivative of the method of Kadan, et al . , J. Virol .. Vol. 66, No. 4, pgs. 2281-2287 (1992) . T47D and SK-BR-3 cells were suspended as follows: Monolayers were rinsed in PBS (Gibco) and incubated in Enzyme Free Cell Dissociation Buffer (Gibco) for 10 minutes at room temperature. Cells were removed from the plate by vigorous agitation.
  • Cell suspensions were triturated briefly with a 1 ml micropipet, and diluted with cell growing media to 10 ml per plate. The cells then filtered through a 50 urn cell strainer (Falcon) , and counted using a hemocytometer. Cells were aliquoted at 2 x 10 5 cells per tube and collected by centrifugation. Cells were resuspended in retroviral supernatant and incubated at room temperature for 1 hour. Cells were collected by centrifugation for 6 seconds and washed once in PBS with 10% goat serum. Cells were resuspended in monoclonal antibody 83A25 directed against C-terminal of gp70 (Evans, et al . , J.
  • the viral supernatants from IV.D. above also were employed in a GP8 cell surface expression assay. Prior to such assay, GP8 cells (Morgan, et al. , 1993) were grown in D10 medium.
  • the viral supernatants of IV.D. above also were used in an NIH3T3 cell titer assay.
  • 3 x 10" NIH3T3 cells were plated to each of the wells in a 6 well titer plate in D10 medium. 12-24 hours later, supernatant from transfection of Psi-2 cells were added to the plate with the Polybrene at a concentration of 8ug/ml. 12-16 hours later, the media was changed to D10/G418 (0.8mg/ml) . Cells were kept in this selection medium for 8-10 days and then stained with 1% Methylene blue in methanol and then counting stained colonies. The assay results are given in Table 1 below.
  • pRSV-1 is a plasmid which contains wild type mouse DHFR cDNA driven by the SV40 promoter, an SV40 poly A sequence, an ampicillin resistance gene, and in the 5' end of the polylinker, there is an RSV LTR promoter.
  • pRSV-1 also is described in Kohli, et al., J.Cell.Phvsiol.. Vol. 142, pgs. 194-200 (1990) .
  • pRSV-1 plasmid was linearized by EcoRV at a polylinker region and dephosphorylated
  • the e23FV-env chimeras in Cee+ based backbone (ChEl, ChE2, and ChE3) were digested with EcoRI
  • chimeras in CAE based backbone ChoAl, ChA2 and ChA3
  • the resultant plasmids were identified by enzyme digestion and named as ChAlRSV, ChA2RSV, ChA3RSV, ChElRSV, ChE2RSV, and ChE3RSV.
  • Chimeras in pRSV-1 are transfected into GPNZ cells (Morgan, et al., 1993) , selected with MTX and amplified. Positive expressing clones are identified by e23 cDNA, PCR priming and immunostaining. Alternatively, chimeras in pRSV-1 are transfected into Psi-2 cells, and subjected to MTX selection and amplification. The cells may be transfected with an appropriate retroviral plasmid vector to produce targeted retroviral particles.
  • the approach of this example was to replace small, disulfide-constrained segments of the ecotropic Moloney murine leukemia virus envelope protein with other receptor- specific ligands.
  • the disulfide-constrained form of the ligands should bind to their specific receptors as well as or better than their corresponding linear form.
  • a constrained form of ⁇ -melanotropin stimulating hormone ( ⁇ - MSH) has been chosen to test this approach.
  • This constrained peptide hormone binds to the target receptors on melanoma cells with at least as high an affinity as the linear form of ⁇ -MSH, and in biological assays has a greater detectable binding than the linear form.
  • One advantage is that there is a simple biological assay system which is very sensitive at detecting ligand binding to receptor.
  • Some human and mouse melanoma tumor cell lines have been found to secrete melanin when stimulated by binding of ⁇ -MSH ligand to the cell- surface MSH receptors. This melanin production can be quantitatively detected using a spectrophotometer which reads an absorbance at 405mm.
  • MSH receptors are present on many melanomas, and the ability to target these tumors with a gene therapy vector may allow for successful treatment of this type of cancer which is rapidly increasing in prevalence. Construction of pcDNA-EF
  • Plasmid pBB2-E was constructed by first amplifying by PCR the coding sequence for the first 262 amino acids of the MoMuLV ecotropic envelope gp 70 protein (i.e., 33aa signal sequence and the first 229 amino acids (i.e., (SEQ ID NO:l)) of the mature protein) from pCEE.
  • oligonucleotides used in this construction were: oligo71 5'-GGAGCTAGCTAGACTGACATGGCGCGTTC-3 ' oligo72 5 ' -CTGTGATCACTATAGATTTTGGTATCTGAGTCG-3 '
  • This PCR product was digested with Nhel and Bell and cloned into the Nhel and BamHI sites of the plasmid pBlueBac2 (Invitrogen Corp., San Diego, CA) to form pBB2-E.
  • the plasmid was modified further by linearizing pBB2-E with BamHI (which cuts at amino acid #222) and inserting two hybridized oligonucleotides (AD298 and AD299) to regenerate amino acids 222-229 as well as a convenient C-terminal fused "FLAG" epitope tag (Kodak/IBI, Rochester, NY) which can be used for facilitating protein purification of this 229 amino acid mature protein.
  • Primers AD298 and AD299 have the following sequences: AD298 5 ' -GATCAGGCTCAGATACCAAAATCTAGACTACAAGGAC
  • pBB2-EF After obtaining the modified plasmid, pBB2-EF, this plasmid was then digested with Hpal and BamHI, to remove the entire coding domain of (SEQ ID NO:l) including the "FLAG" tag, and such fragment was cloned into pCEE+ to construct the intermediary construct pCEE+F.
  • pCEE+F was digested with EcoRI and NotI to remove the entire coding region of (SEQ ID NO:1) /FLAG tag fusion protein and such fragment was cloned into EcoRI and NotI sites of pcDNA3 (Invitrogen Corp., San Diego, CA) , thereby generating pcDNA-EF.
  • alpha- MSH ⁇ -Melanotropin Stimulating Hormone
  • MSH ⁇ -Melanotropin Stimulating Hormone
  • MuLV ⁇ -Melanotropin Stimulating Hormone
  • the oligonucleotides used in the construction of these alpha- MSH/MuLV chimeric proteins and the location of replaced amino acids are indicated below:
  • PCR reactions involved the amplification of primer combinations AD740+AF40 , AD740+AF45 , AD738+AF39 , and AD738+AF44 in individual reactions consisting of PCR buffer ( final concentration : 50mM KC1 , lOmM Tris -HCI , pH8 . 3 , 1 . 5mM MgCl 2 , and 0 . 001% gelatin) , 0 .2mM dNTPs , 80mM additional MgCl 2 , 0 . 5 units Taq polymerase , 50 ng of pcDNA-EF plasmid, and 12 .
  • PCR buffer final concentration : 50mM KC1 , lOmM Tris -HCI , pH8 . 3 , 1 . 5mM MgCl 2 , and 0 . 001% gelatin
  • 0 .2mM dNTPs 80mM additional MgCl 2 , 0
  • the PCR products were joined [ (AD740+AF40) + (AD738+AF39) and (AD740+AF45) + (AD738+AF44) ] and amplified in reactions similar to that detailed above, except no pcDNA-EF plasmid was added to the reactions, and the only oligonucleotides added to the reactions were AD738 and AD740 at the above indicated concentrations.
  • a small portion of the PCR products were again identified using agarose gel electrophoresis/ethidium bromide staining.
  • PCR products were extracted with phenol/chloroform/isoamyl alcohol (25:24:1) , back extracted with 1 mM Tris-HCI (pH8.0)/l mM EDTA, and the aqueous phase precipitated by addition of 0.1 volume of 3M sodium acetate, pH 4.6 and 2.5 volumes of 100% ethanol.
  • the PCR products were digested with restriction enzymes BstEII (New England Biolabs, Beverly, MA) and AccIII (Promega, Madison, WI) , purified using agarose gel electrophoresis as above, and subsequently ligated into the BstEII and AccIII sites of the pcDNA-EF plasmid.
  • Frozen competent XLl-blue E. coli (Stratagene, La Jolla, California) were transformed with the ligation products according to the method of Hanahan, J. Mol. Biol., Vol. 166, pgs. 557-580 (1983) .
  • the amino acid replacement plasmids are p3-l and p3-2, and replace residues S74-S91 with the residues A-H-F-R-W-C-K-A-V-C-E-H-F- R-W-G-K-A and E-H-F-R-W-C-K-A-V-C-E-H-F-R-W-G-K-A, respectively.
  • the amino acid replacement plasmid p6-3 replaces residues G80-P88 with S-C-A-H-F-R-W-C-K-P-V.
  • C0S7 cells were maintained in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 2mM glutamine at 37°C and 5% C0 2 .
  • Plasmid DNA from p3-l, p3-2, or p6-3 was prepared for transfection from overnight cultures with a Plasmid Maxi Kit (Qiagen, Chatsworth, CA) .
  • C0S7 cells were plated on 100mm tissue culture dishes at a concentration of 6 x 10 5 cells/lOOmm dish. Twelve to sixteen hours after plating the cells, the media in the dish was aspirated and 9ml of fresh media was added to the cells. The cells were then returned to the incubator at 37°C/5% CO-.
  • the cells were transfected with 30 ⁇ g plasmid DNA by the calcium phosphate method (Graham, F.L. and A.J. van der Eb (1973) Virology 52, 456-467) .
  • the media was aspirated, cells were washed with 10ml of Dulbecco's phosphate-buffered saline, and fresh media was added to the cells.
  • the media was aspirated from the plates and replaced with DMEM + 1% fetal bovine serum + 2 mM glutamine containing 600 ⁇ g/ml G418 for selection of COS7 cells.
  • B16-F1 mouse melanoma cells were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum and 2mM glutamine at 37°C and 5% CO-. These B16-F1 cells secrete melanin when stimulated by the binding of alpha-MSH to the cell-surface receptors. The melanin which is secreted can be measured spectrophotometrically at an absorbance of 405nm.
  • the B16-F1 cells were plated in a 96-well microtiter plate at a concentration of 2500 cells/well. Approximately 24 hours after plating the cells, the media was aspirated and either media which contained known concentrations of alpha-MSH ligand (Sigma, St.
  • these plasmids are prepared for transfection into the pre-producer cell line, GPL.
  • the chimeric ⁇ MSH/MuLV envelope protein plasmids are cotransfected with the plasmid pPUR (Clonetech, Palo Alto, CA) at a ratio of 29:1 to allow for selection of stable producer clones with the antibiotic puromycin. After selection of stable clones, the best producer clone is identified by screening for envelope protein expression on the cell surface, as well as virus binding, fusion, and transduction of MSH-specific target cells.
  • GENERAL INFORMATION (i) APPLICANT: Anderso , W. French Zhao, Yi Chiang, Yawen MacKrell, Albert Januszeski, Michael
  • ADDRESSEE Carella, Byrne, Bain, Gilfillan, Cecchi, Stewart & Olstein
  • NAME/KEY Receptor binding region of ecotropic gp70 protein
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:l:
  • Trp Asp Phe lie Thr Val Asn Asn Asn Leu
  • NAME/KEY Receptor binding region of amphotropic gp 70 protein
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 2: Val Gly Met Ala Glu Ser Pro His Gin Val
  • NAME/KEY Receptor binding region of 10A1 murine leukemia virus envelope (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Val Gly Met Ala Glu Ser Pro His Gin Val
  • NAME/KEY Receptor binding region of xenotropic murine leukemia virus .
  • NAME/KEY Receptor binding region of polytropic MX27 provirus .
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 5: Val Ser Val Gin His Asp Ser Pro His Gin
  • NAME/KEY polynucleotide encoding receptor binding region of ecotropic gp 70 protein
  • TGT TCC AGA GAC TGC GAA GAA CCT TTA ACC TCC CTC ACC CCT CGG TGC AAC ACT GCC TGG 300
  • MOLECULE TYPE polynucleotide
  • TGT GGT AAA TGG GGG TGT GAA ACC ACC GGA
  • NAME/KEY polynucleotide encoding receptor binding region of xenotropic murine leukemia virus
  • NAME/KEY polynucleotide encoding receptor binding region of polytropic MX 27 provirus
  • GCG GGC AAA AAG GCC AGC TGG GAT GGC CCC

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Abstract

La présente invention décrit une particule vecteur rétrovirale possédant une enveloppe polypeptidique modifiée dans laquelle une partie de la région de liaison du récepteur de l'enveloppe est remplacée par un polypeptide de ciblage qui se lie à un ligand ou à un récepteur sur les cellules ciblées. Le polypeptide de ciblage peut être un anticorps à chaîne simple. De telles particules vecteurs rétrovirales servent à amener des agents thérapeutiques codant des gènes vers des cellules ou des tissus ciblés in vivo.
PCT/US1996/003908 1995-03-24 1996-03-22 Polypeptide d'enveloppe virale modifie WO1996030504A1 (fr)

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AU53202/96A AU5320296A (en) 1995-03-24 1996-03-22 Modified viral envelope polypeptide

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US08/409,648 1995-03-24

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997030169A1 (fr) * 1996-02-13 1997-08-21 Fred Hutchinson Cancer Research Center Cellules d'encapsidation de vecteurs retroviraux 10a1 et utilisations de ce type de cellules
WO1997032032A1 (fr) * 1996-03-01 1997-09-04 The Regents Of The University Of California PROCEDES DE CRIBLAGE DESTINES A DETECTER DES CIBLES ARNm D'ENZYMES DE MISE EN FORME
WO1998044938A1 (fr) * 1997-04-10 1998-10-15 University Of Southern California Proteines modifiees se fixant a des composants de matrice extracellulaires
WO1999028488A3 (fr) * 1997-11-28 1999-08-12 Bundesrepublik Deutschland Let Vecteurs retroviraux pseudotypes a proteines de surface d'enveloppe et procede de fabrication desdits vecteurs pour le transfert selectif de genes
US6004798A (en) * 1997-05-14 1999-12-21 University Of Southern California Retroviral envelopes having modified hypervariable polyproline regions
EP1007716A4 (fr) * 1997-04-17 2000-06-14 Univ California Utilisation de vecteurs lentiviraux pour l'introduction d'un antigene dans des cellules dendritiques
US7078483B2 (en) 1998-04-29 2006-07-18 University Of Southern California Retroviral vectors including modified envelope escort proteins
US7276488B2 (en) 1997-06-04 2007-10-02 Oxford Biomedica (Uk) Limited Vector system
US7514546B2 (en) 1999-11-18 2009-04-07 Oxford Biomedica (Uk) Ltd. Antibodies
US7531648B2 (en) 1997-06-04 2009-05-12 Oxford Biomedica (Uk) Limited Vector encoding an antibody that binds 5T4 antigen
US7531322B2 (en) 1997-11-28 2009-05-12 Bundesrepublik Deutschland Cell-specific retroviral vectors with antibody domains and method for the production thereof for selective gene transfer
US8052966B2 (en) 2003-04-21 2011-11-08 University Of Southern California Methods and compositions for treating metastatic cancer
US8828378B2 (en) 2000-11-29 2014-09-09 The University Of Southern California Targeted vectors for cancer immunotherapy
US9017659B2 (en) 2006-11-03 2015-04-28 Epeius Biotechnologies Corporation Pathotropic targeted gene delivery system for cancer and other disorders

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* Cited by examiner, † Cited by third party
Title
BONE MARROW TRANSPLANTATION, 1992, Vol. 9, Suppl. 1, ETIENNE-JULAN et al., "Cell Targeting by Murine Recombinant Retroviruses", pages 139-142. *
FASEB J., February 1995, Vol. 9, MILLER et al., "Targeted Vectors for Gene Therapy", pages 190-199. *
J. VIROLOGY, August 1993, Vol. 67, No. 8, MORGAN et al., "Analysis of the Functional and Host Range-Determining Regions of the Murine Ecotropic and Amphotropic Retrovirus Envelope Proteins", pages 4712-4721. *
SCIENCE, 25 November 1994, Vol. 266, KASAHARA et al., "Tissue-Specific Targeting of Retroviral Vectors Through Ligand-Receptor Interactions", pages 1373-1376. *

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5766945A (en) * 1996-02-13 1998-06-16 Fred Hutchinson Cancer Research Center 10A1 Retroviral packaging cells and uses thereof
WO1997030169A1 (fr) * 1996-02-13 1997-08-21 Fred Hutchinson Cancer Research Center Cellules d'encapsidation de vecteurs retroviraux 10a1 et utilisations de ce type de cellules
US5866333A (en) * 1996-03-01 1999-02-02 Regents Of The University Of California Screening methods to detect mRNA targets of editing enzymes
WO1997032032A1 (fr) * 1996-03-01 1997-09-04 The Regents Of The University Of California PROCEDES DE CRIBLAGE DESTINES A DETECTER DES CIBLES ARNm D'ENZYMES DE MISE EN FORME
US8530441B2 (en) 1997-04-10 2013-09-10 University Of Southern California Transgene delivering retrovirus targeting collagen exposed at site of tissue injury
US8871734B2 (en) 1997-04-10 2014-10-28 The University Of Southern California Transgene delivering retrovirus targeting collagen exposed at site of tissue injury
EP0973538A4 (fr) * 1997-04-10 2001-09-19 Univ Southern California Proteines modifiees se fixant a des composants de matrice extracellulaires
US7820157B2 (en) 1997-04-10 2010-10-26 University Of Southern California Transgene delivering retrovirus targeting collagen exposed at site of tissue injury
US6864082B2 (en) 1997-04-10 2005-03-08 University Of Southern California Modified viral surface proteins for binding to extracellular matrix components
US8148509B2 (en) 1997-04-10 2012-04-03 University Of Southern California Transgene delivering retrovirus targeting collagen exposed at site of tissue injury
WO1998044938A1 (fr) * 1997-04-10 1998-10-15 University Of Southern California Proteines modifiees se fixant a des composants de matrice extracellulaires
US7347998B2 (en) 1997-04-10 2008-03-25 University Of Southern California Method of delivering therapeutic agents to site of tissue injury
EP2008664A3 (fr) * 1997-04-10 2009-03-11 University of Southern California Protéines modifiées qui lient des composants de la matrice extracellulaire
EP1007716A4 (fr) * 1997-04-17 2000-06-14 Univ California Utilisation de vecteurs lentiviraux pour l'introduction d'un antigene dans des cellules dendritiques
US6004798A (en) * 1997-05-14 1999-12-21 University Of Southern California Retroviral envelopes having modified hypervariable polyproline regions
US7276488B2 (en) 1997-06-04 2007-10-02 Oxford Biomedica (Uk) Limited Vector system
US7531648B2 (en) 1997-06-04 2009-05-12 Oxford Biomedica (Uk) Limited Vector encoding an antibody that binds 5T4 antigen
US7718627B2 (en) 1997-06-04 2010-05-18 Oxford Biomedica (Uk) Limited Vector
US8084249B2 (en) 1997-06-04 2011-12-27 Oxford Biomedica (Uk) Limited Vector
US7531322B2 (en) 1997-11-28 2009-05-12 Bundesrepublik Deutschland Cell-specific retroviral vectors with antibody domains and method for the production thereof for selective gene transfer
US6544779B1 (en) 1997-11-28 2003-04-08 Bundesrepublik Deutschland Pseudo-type retroviral vectors with modifiable surface capsid proteins
WO1999028488A3 (fr) * 1997-11-28 1999-08-12 Bundesrepublik Deutschland Let Vecteurs retroviraux pseudotypes a proteines de surface d'enveloppe et procede de fabrication desdits vecteurs pour le transfert selectif de genes
US7078483B2 (en) 1998-04-29 2006-07-18 University Of Southern California Retroviral vectors including modified envelope escort proteins
US7514546B2 (en) 1999-11-18 2009-04-07 Oxford Biomedica (Uk) Ltd. Antibodies
US8828378B2 (en) 2000-11-29 2014-09-09 The University Of Southern California Targeted vectors for cancer immunotherapy
US8052966B2 (en) 2003-04-21 2011-11-08 University Of Southern California Methods and compositions for treating metastatic cancer
US9017659B2 (en) 2006-11-03 2015-04-28 Epeius Biotechnologies Corporation Pathotropic targeted gene delivery system for cancer and other disorders

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