WO1997023608A1 - Compositions et procedes permettant de doter des vecteurs d'acheminement de genes d'elements de ciblage lies de maniere covalente - Google Patents

Compositions et procedes permettant de doter des vecteurs d'acheminement de genes d'elements de ciblage lies de maniere covalente Download PDF

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WO1997023608A1
WO1997023608A1 PCT/US1996/020543 US9620543W WO9723608A1 WO 1997023608 A1 WO1997023608 A1 WO 1997023608A1 US 9620543 W US9620543 W US 9620543W WO 9723608 A1 WO9723608 A1 WO 9723608A1
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Prior art keywords
linking agent
gene delivery
delivery vehicle
targeting
gdv
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PCT/US1996/020543
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English (en)
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Margaret D. Moore
James G. Respess
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Chiron Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers

Definitions

  • the present invention relates generally to compositions and methods for targeting gene delivery vehicles to one or more specific cell or tissue types. Specifically, the invention concerns compositions and methods which utilize targeting elements covalently bound to the surface of a gene delivery vehicle via a multifunctional linking agent.
  • a recombinant retroviral vector expressing HIV gp 160/120 envelope proteins was shown to elicit a cytotoxic T-cell response against cells infected with HIV in Warner et al , (AIDS Res. Hum. Retro. 7:645, 1991).
  • a recombinant retroviral vector expressing the hypoxanthine phosphorelase transferase (HPRT) gene was transduced into bone marrow cells and shown to produce adequate levels of HPRT to correct the metabolic defect, known as Lesch-Nyhan syndrome, in culture.
  • HPRT hypoxanthine phosphorelase transferase
  • one difficulty with recombinant retroviruses and other gene delivery vehicles is that they are difficult to target to a selected cell type or tissue where it is desired to affect treatment.
  • the present invention provides compositions and methods for targeting gene delivery vehicles to one or more specific cell or tissue types. It is the object ofthe present invention to provide a gene delivery vehicle to which a targeting element is covalently bound by a linking agent such that the targeting element is capable of interacting with a molecule present on the surface of a target cell.
  • the targeting element is covalently bound to the surface ofthe gene delivery vehicle by a multifunctional linking agent.
  • the multifunctional linking agent is selected from the group consisting of a homobifunctional linking agent, a heterobifunctional linking agent and a trifunctional linking agent.
  • the multifunctional linking agent may be selected from the group consisting of 4(4-N-maleimidophenyl)butyric acid hydride*HCl*l/2 dioxane (MPBH), succinimidyl 4-(N-maleimidomethyl)cyclohexane-l- carboxylate (SMCC), sulfosuccinimidyl (4(azidosalicylamido)hexanoate (Sulfo-NHS-LC- ASA), sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl- l,3'-dithiopropionate (Sulfo-SBED), and disuccinimidyl suberate (DSS), N-succinimidyl-3- (2-pyridyldithio)-propionate (SPDP)), sulf
  • MPBH
  • the targeting element is covalently bound to a gene delivery vehicle by a multifunctional linking agent further comprising a carbohydrate linking agent.
  • the carbohydrate ofthe carbohydrate linking agent is a monosaccharide, a disaccharide, or an oligosaccharide.
  • the carbohydrate is a monosaccharide
  • the monosaccharide is preferably selected from the group consisting of fructosamine, glucosamine, galactosamine, and mannosamine.
  • the carbohydrate is a disaccharide
  • the disaccharide is preferably selected from the group consisting of aminated sucrose, animated maltose, aminated trehalose, and aminated lactose.
  • the oligosaccharide is preferably selected from the group consisting of N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, aminated N- acetylmuramic acid, and aminated N-acetyl-D-neuraminic acid.
  • any gene delivery vehicle is suitable for uses in this invention.
  • Preferred gene delivery vehicles include recombinant viral vectors, nucleic acids associated with liposomes, nucleic acids associated with polycations, modified bacteriophage, and bacteria.
  • a recombinant viral vector is utilized, preferably it is a recombinant virus derived from a virus selected from the group consisting of adenovirus, astrovirus, coronavirus, hepadnevirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, and poxvirus.
  • the recombinant viral vector is a togavirus or a retrovirus.
  • the alphavirus is preferably selected from the group consisting of Sindbis virus, Semliki Forest virus, Middleberg virus, Ross River virus, and Venezuelan equine encephalitis virus
  • the retrovirus is preferably selected from the group consisting of avian leukosis virus, bovine leukemia virus, murine leukemia virus, mink-cell focus-inducing virus, murine sarcoma virus, reticuloendotheliosis virus, gibbon ape leukemia virus, Mason-Pfizer monkey virus, rous sarcoma virus, baboon endogenous virus, endogenous feline retrovirus (e.g., RDl 14), and mouse or rat gL30 sequences used as a retroviral vector.
  • the retrovirus is a murine leukemia retrovirus
  • the virus is preferably selected from the group consisting of Abelson, Friend, Graffi, Kristen, Rauscher, and Moloney leukemia retrovirus, with the latter being particularly preferred. It is preferable that the recombinant viral vector is replication defective.
  • the gene delivery vehicle further comprises a fusagenic protein.
  • fusagenic proteins include those selected from the group consisting of ecotropic murine retrovirus envelope proteins, herpes simplex virus gH fusagenic proteins, he ⁇ es simplex virus gL fusagenic proteins, Epstein-Barr fusagenic proteins, measles virus fusagenic proteins, and malarial sporozoite fusagenic proteins.
  • suitable targeting elements include proteins, peptides, carbohydrates, and small molecules which specifically interact with a molecule, e.g., a receptor, present on the surface ofthe cell or tissue type(s) to be targeted.
  • the targeting element employed is a protein.
  • the protein-based targeting element is selected from the group consisting of a receptor, a ligand, and an antibody (or antigen binding domain thereof).
  • the targeting element is a receptor, the receptor is preferably selected from the group consisting of CD4, CD8, CD21, and fimbriae.
  • the targeting element is a ligand
  • the ligand is preferably selected from a cytokine, lymphokine, polypeptide hormone, peptide or nonprotein molecule.
  • Representative cytokines include IL-l type II, IL-2b, IL-3, IL-6, IL-7, IL-8, IL- 10, and IL-l 2.
  • Representative lymphokines include GM-CSF, G-CSF, M-CSF, SCF, and the flk-2 ligand.
  • Representative polypeptide hormones include FSH, GH, luteinizing hormone, MSH, erythropoietin, nerve growth factor, VEGF, UP A, and epidermal growth factor.
  • polypeptides include neuromedin, insulin, transferrin, asialoglycoprotein, lectin, and collagen and a representative nonprotein molecule is LDL.
  • the targeting element is a monoclonal antibody
  • the monoclonal antibody is preferably selected from the group consisting of 12.8, MylO, HPCA-2, anti-CD8, 4D5,
  • target cells are disease associated, e.g., neoplastic cells, autoimmune cells, or cells infected with a viral or bacterial pathogen.
  • targeted cells are cells which normally express, or do not express, as the case may be, a particular gene product in an appropriate physiological amount, for example, b-islet cells in the pancreas which produce insulin in non-diabetic animals, pituitary cells which express growth hormone, and hematopoietic tissue which expresses ADA.
  • compositions which comprise a targeted gene delivery vehicle in a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is lyophilized, wherein the targeted gene delivery vehicle upon reconstitution is suitable for administration to animals.
  • compositions described herein comprise mixing a gene delivery vehicle and a targeting element in the presence of a multifunctional linking agent under conditions which allow the multifunctional linking agent to become covalently bound to both the gene delivery vehicle and targeting element(s).
  • Still other aspects ofthe invention concern methods for administering a therapeutically effective amount ofthe targeted gene delivery vehicles.
  • targeted gene delivery vehicles are administered to animals in order to treat disease.
  • Other emboidments concern the prophylactic use of targeted gene delivery vehicles in animals.
  • Representative diseases which may be treated using targeted gene delivery vehicles include diseases selected from the group consisting of an infectious disease, cancer, a genetic disease, an autoimmune disease, and a cardiovascular disease.
  • the present invention provides gene delivery vehicles (GDVs) to which targeting elements have been covalently bound to the exterior surface of he GDV by a multifunctional linking agent such that the GDV is be targeted to a selected cell or tissue type.
  • GDVs gene delivery vehicles
  • a gene delivery vehicle is a composition capable of delivering a nucleic acid molecule to a eukaryotic cell.
  • Representative examples of gene delivery vehicles include recombinant viral vectors (e.g., retroviruses; see WO 89/09271, and alphaviruses such as Sindbis; see WO 95/07994), other recombinant and non-recombinant viral systems (e.g., adenovirus; see WO 93/19191), nucleic acid molecules associated with one or more condensing agents (see WO 93/03709), nucleic acid molecules associated with liposomes (Wang, et al, PNAS 84:7%51, 1987), modified bacteriophage, or bacteria.
  • recombinant viral vectors e.g., retroviruses; see WO 89/09271, and alphaviruses such as Sindbis; see WO 95/07994
  • the GDV carries a nucleic acid molecule to be transferred to a target cell.
  • the nucleic acid molecule may itself exhibit biological activity which directly effects the target cell (e.g., a ribozyme, antisense RNA, etc.)
  • the nucleic acid molecule may encode a desired substance such as a protein, (e.g., an enzyme or an antibody) and/or a nucleic acid having biological activity which, once expressed, will affect the target cell.
  • a desired substance such as a protein, (e.g., an enzyme or an antibody) and/or a nucleic acid having biological activity which, once expressed, will affect the target cell.
  • nucleic acids which themselves have biological activity include an antisense nucleic acid molecules and ribozymes.
  • the GDV is a recombinant viral vector derived from a virus such as an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, retrovirus, togavirus or adenovirus.
  • the recombinant viral vector is a recombinant retroviral vector.
  • Retroviral GDVs may be readily constructed from a wide variety of retroviruses, including for example, B, C, and D type retroviruses, as well as spumaviruses and lentiviruses (see RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985).
  • retroviruses include those discussed in RNA Tumor Viruses, supra, as well as a variety of xenotropic retroviruses (e.g., NZB-X1, NZB-X2 and NZB9.] (see O'Neill et al, J. Vir. 53: 100, 1985)) and polytropic retroviruses (e.g., MCF and MCF-MLV (see Kelly et al, J. Vir. 45( ⁇ ):29l, 1983)).
  • xenotropic retroviruses e.g., NZB-X1, NZB-X2 and NZB9.
  • polytropic retroviruses e.g., MCF and MCF-MLV (see Kelly et al, J. Vir. 45( ⁇ ):29l, 1983)
  • retroviruses may be readily obtained from depositories or collections such as the American Type Culture Collection (ATCC, Rockville, MD), or isolated from known sources using commonly available techniques.
  • retroviral GDVs which may be utilized in practicing the present invention are described in U.S. Patent Nos. 5,219,740 and 4,777,127, EP 345,242 and WO 91/02805.
  • retroviruses which include avian leukosis virus (ATCC Nos. VR-535 and VR-247), bovine leukemia virus (VR-1315), murine leukemia virus (MLV), mink-cell focus-inducing virus (Koch et al, J. Vir. 49:828, 1984; and Oliff et al, J. Vir. 48:542, 1983), murine sarcoma virus (ATCC Nos.
  • VR-844, 45010 and 45016 reticuloendotheliosis virus (ATCC Nos VR-994, VR-770 and 4501 1), rous sarcoma virus, Mason-Pfizer monkey virus, baboon endogenous virus, endogenous feline retrovirus (e.g., RD1 14), and mouse or rat gL30 sequences used as a retroviral vector.
  • Particularly preferred strains of MLV from which recombinant retroviruses can be generated include 4070A and 1504A (Hartley and Rowe, J. Vir. 19:19, 1976), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi (Ru et al. , J. Vir. 67:4722, 1993 ; and
  • a particularly preferred non-mouse retrovirus is rous sarcoma virus.
  • Preferred rous sarcoma viruses include Bratislava (Manly et al , J. Vir.
  • retroviral GDVs Any of the above retroviruses may be readily utilized in order to assemble or construct retroviral GDVs given the disclosure provided herein and standard recombinant techniques (e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989 and Kunkle, PNAS 52:488, 1985) known in the art.
  • portions ofthe retroviral GDVs may be derived from different retroviruses.
  • recombinant retroviral vector LTRs may be derived from a murine sarcoma virus, a tRNA binding site from a rous sarcoma virus, a packaging signal from a MLV, and an origin of second strand synthesis from an avian leukosis virus.
  • These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see U.S. Patent No. 5,591,624, issued January 1, 1996).
  • recombinant retroviruses can be produced which direct the site-specific integration ofthe recombinant retroviral genome into specific regions of the host cell DNA.
  • a retroviral vector construct should include a 5' LTR, a tRNA binding site, a packaging signal, a nucleic acid molecule encoding one or more genes of interest, an origin of second strand DNA synthesis, and a 3' LTR.
  • a retroviral vector construct may also include transcriptional promoter/enhancer or locus defining element(s), or other elements which control gene expression by means such as alternate splicing, nuclear RNA export, post-translational modification of messenger, or post-transcriptional modification of protein.
  • a retroviral vector construct may also include one or more selectable markers that confer resistance of vector transduced or transfected cells to thymidine kinase (TK), hygromycin, phleomycin, histidinol, or dihydrofolate reductase (DHFR), as well as one or more specific restriction sites and a translation termination sequence.
  • TK thymidine kinase
  • hygromycin hygromycin
  • phleomycin histidinol
  • DHFR dihydrofolate reductase
  • the GDV is derived from a togavirus.
  • Preferred togaviruses include alphaviruses, in particular, those described in WO 95/07994 filed September 15, 1994.
  • Sindbis viral vectors typically comprise a 5' sequence capable of initiating Sindbis virus transcription, a nucleotide sequence encoding Sindbis non-structural proteins, a viral junction region inactivated so as to prevent subgenomic fragment transcription, and a Sindbis RNA polymerase recognition sequence.
  • the viral junction region may be modified so that subgenomic fragment transcription is reduced, increased, or maintained.
  • an alphavirus-derived GDV may comprise a first viral junction region which has been inactivated in order to prevent transcription ofthe subgenomic fragment and a second viral junction region which has been modified such that subgenomic fragment transcription is reduced.
  • an alphavirus-derived GDV may also include a 5' promoter capable of initiating synthesis of viral RNA from cDN A and a 3' sequence which controls transcription termination.
  • the recombinant alphaviral vectors do not encode structural proteins and the nucleic acid molecule may be located downstream from the viral junction region.
  • the nucleic acid molecule encoding the gene(s) of interest may be located downstream from the second viral junction region.
  • the vector construct may further comprise a polylinker located between the viral junction region and the nucleic acid molecule.
  • the polylinker does not contain restriction sites found in the corresponding naturally occuring alphavirus or recombinant vector backbones made therefrom.
  • togaviral vectors that may be utilized in the present invention include those derived from Semliki Forest virus (ATCC VR-67; ATCC VR-1247),
  • Sindbis vector constructs may be readily prepared essentially as described in WO95/07994.
  • the recombinant viral vector may be a recombinant adenoviral vector.
  • adenoviral vector Such vectors may be readily prepared and utilized given the disclosure provided herein (see Berkner, Biotechniques 6:616, 1988, and Rosenfeld et al, Science 252:431, 1991, WO 93/07283, WO 93/06223, and WO 93/07282).
  • Other viral vectors suitable for use in the present invention include, for example, those derived from poliovirus (Evans et al, Nature 339:385, 1989, and Sabin et al, J. Biol. Standardization 7:115, 1973) (ATCC VR-58); rhinovirus (Arnold et al, J. Cell. Biochem.
  • the GDV comprises a nucleic acid molecule associated with a condensing agent (e.g., polycations).
  • a condensing agent e.g., polycations. Polycations condense the nucleic acid molecule by masking the negatively charged phosphate backbone, permitting the molecule to fold into a more compact form.
  • the GDV is a nucleic acid molecule associated with a liposome.
  • Liposomes are small, lipid vesicles comprised of an aqueous compartment enclosed by a lipid bilayer, typically spherical or slightly elongated structures several hundred Angstroms in diameter.
  • a liposome can fuse with the plasma membrane of a cell or with the membrane of an endocytic vesicle within a cell which has internalized the liposome, thereby releasing its contents into the cytoplasm.
  • the liposome membrane Prior to interaction with the surface ofa cell, however, the liposome membrane acts as a relatively impermeable barrier which sequesters and protects its contents, for example, from degradative enzymes in the plasma.
  • Liposomes can encapsulate a variety of nucleic acid molecules including DNA, RNA, plasmids, and vectors such those described in the present invention.
  • the GDV is a modified bacteriophage which can deliver therapeutic nucleic acid molecules to eukaryotic cells.
  • One such representative bacteriophage system (based on bacteriophage lambda) is described in co-owned U.S.S.N. 08/366,522, filed December 30, 1994.
  • the only lambda nucleotide sequences contained in the nucleic acid molecule of such a lambda-based system are two cos sites, one at the 5' and 3' ends ofthe linear DNA to be packaged, leaving up to about 50 Kb available for therapeutic gene(s) or other sequences.
  • the GDV is a bacterial cell comprising a nucleic acid molecule for delivery to eukaryotic cells.
  • the bacterial cell may express and present a cytotoxic agent, such as an anti-tumor agent, on its surface or, alternatively, secrete it into the surrounding medium.
  • Representative examples of bacterial cell GDVs include BCG (Stover, Nature 351:456, 1991) and Salmonella (Newton et al, Science 244:70, 1989).
  • a targeted GDV may carry a eukaryotic layered vector initiation system or other nucleic acid expression systems. See WO 95/07994 for additional details in the construction of such systems.
  • GDVs useful in the practice of this invention above may include or contain one or more nucleic acid molecules.
  • a wide variety of nucleic acid molecules may be utilized within the context ofthe present invention, including, for example, those which themselves have biological activity or which encode gene products (e.g., proteins, anti-sense RNAs, and ribozymes, among others).
  • the GDVs ofthe invention can contain a variety of nulceic acid sequences of therapeutic interest. See e.g., WO 91/02805, WO 95/07994, WO 96/20414 and U.S. Patent Nos. 5,399,346, 5,580,859, 5,192,553 for a description of such nucleic acid sequences.
  • a targeted GDV may deliver a ribozyme directly to the target cell.
  • a GDV may deliver a nucleic acid molecule which encodes one or more ribozymes (Haseloff and Gerlach, Nature 334:585, 1989).
  • the nucleic acid molecule encodes one or more proteins.
  • Representative proteins which may be encoded by a nucleic acid molecule include, for example, receptors, cytotoxins, immunomodulatory factors (e.g., lymphokines and cytokines), immunoreactive proteins (e.g., inhibitory, immunogenic and immunosuppressive polypeptides) and replacement proteins (e.g., polypeptide hormones and enzymes expressed at insufficient levels in patients' suffering from the corresponding disease).
  • receptors many are involved in cell growth, either by monitoring the external environment and signaling the cell to respond appropriately. Other receptors are intracellular in nature. If either the monitoring or signaling mechanisms fail, the cell will no longer respond appropriately to particular signals and may therefore exhibit uncontrolled or aberrant growth.
  • receptors or receptor-like structures may function as altered cellular components, including, for example, neu (also referred to as the human epidermal growth factor receptor (HER) Slamon et al, Science 244:707, 1989; Slamon et al , Cancer Cells 7:371 , 1989; Shih et al , Nature 290:261 , 1981 Schechter, Nature
  • neu also referred to as the human epidermal growth factor receptor (HER) Slamon et al, Science 244:707, 1989; Slamon et al , Cancer Cells 7:371 , 1989
  • Shih et al Nature 290:261 , 1981 Schechter, Nature
  • the targeted GDV contains a nucleic acid molecule encoding a product which is not itself toxic, but when processed or modified by a protein, such as a protease specific to a viral or other pathogen, is converted into a toxic form.
  • a GDV may carry a nucleic acid molecule encoding a proprotein which becomes toxic upon processing by a viral, e.g., HIV, protease.
  • a proprotein which becomes toxic upon processing by a viral, e.g., HIV, protease.
  • an engineered inactive proprotein form ofthe toxic ricin or diphtheria A chain can be cleaved to the active form by arranging for a virally encoded protease to recognize and cleave the "pro" element (see WO 95/14091).
  • nucleic acid molecules which express one or more gene products capable of activating an otherwise inactive precursor into an active inhibitor of a pathogenic agent, or a conditional toxic palliative, i.e., palliatives that are toxic for the cell expressing the pathogenic condition.
  • inactive precursors may be converted into active inhibitors of a pathogenic agent.
  • antiviral nucieoside analogs such as AZT or ddC are metabolized by cellular mechanisms to a nucleotide triphosphate form in order to specifically inhibit retroviral reverse transcriptase and thus inhibit viral replication (Furmam et al, Proc. Natl Acad. Sci. USA 53:8333-8337, 1986).
  • GDVs which comprise a nucleic acid molecule which encodes a substance (e.g., a protein) such as he ⁇ es simplex virus thymidine kinase (HSVTK), Varicella Zoster virus thymidine kinase (VZVTK), or other such "pro-drug activating enzymes" which selectively monophosphorylate certain purine arabinosides and substituted pyrimidine compounds (e.g., AZT or ddC), converting them to cytotoxic or cytostatic metabolites, are particularly useful.
  • a substance e.g., a protein
  • HSVTK simplex virus thymidine kinase
  • VZVTK Varicella Zoster virus thymidine kinase
  • pro-drug activating enzymes which selectively monophosphorylate certain purine arabinosides and substituted pyrimidine compounds (e.g., AZT or ddC), converting them to
  • GDVs may be utilized to express a pro ⁇ drug activating enzyme in a target cell which can be later destroyed by exposure to the appropriate "pro-drug", (e.g., gancyclovir, acyclovir, or any of their analogs (e.g., FIAU, FIAC, DHPG)) which is then phosphorylated into its corresponding active nucleotide triphosphate form.
  • a pro-drug e.g., gancyclovir, acyclovir, or any of their analogs (e.g., FIAU, FIAC, DHPG)
  • a nucleic acid molecule may code for a protein which performs phosphorylation, phosphoribosylation, ribosylation, or other metabolism of a purine- or pyrimidine-based drug.
  • Such nucleic acid molecules may have no equivalent in mammalian cells, and may be derived from organisms such as a virus, bacterium, fungus, or protozoan.
  • Representative examples include nucleic acid molecules which encode: E. coli guanine phosphoribosyl transferase ("gpt"), which converts thioxanthine into thioxanthine monophosphate (see Besnard et al, Mol Cell Biol.
  • alkaline phosphatase which converts inactive phosphorylated compounds such as mitomycin phosphate and doxorubicin-phosphate to toxic dephosphorylated compounds
  • fungal e.g., Fusarium oxysporum
  • bacterial cytosine deaminase which converts 5- fluorocytosine to 5-fluorouracil
  • carboxypeptidase G2 which cleaves glutamic acid from para-N-bis (2-chloroethyl) aminobenzoyl glutamic acid, to create a toxic benzoic acid mustard
  • Penicillin- V amidase which converts phenoxyacetamide derivatives of doxorubicin and melphalan to toxic compounds.
  • Conditionally lethal products have application to many presently known purine- or pyrimidine-based anticancer drugs, which often require intracellular ribosylation or phosphorylation to become effective cytotoxic agents.
  • the conditionally lethal gene product could also metabolize a nontoxic drug which is not a purine or pyrimidine analog to a cytotoxic form (see Searle et al., Brit. J. Cancer 53:377, 1986).
  • the nucleic acid molecule carried by the targeted GDV may direct the expression of one or more immunomodulatory factors.
  • An immunomodulatory factor is one which, when expressed by one or more ofthe cells involved in an immune response, or which, when added exogenously to the cells, causes an immune response to be different in quality or potency from that which would have occurred in the absence of the factor.
  • the immunomodulatory factor may also be expressed from an endogenous gene whose expression is driven or controlled by a gene product encoded by the nucleic acid molecule.
  • the quality or potency of a response may be measured by a variety of known assays, for example, in vitro assays which measure cellular proliferation (e.g., ⁇ H thymidine uptake), and in vitro cytotoxic assays (e.g., which measure 51 Cr release) (see, Warner et al, AIDS Res. and Human Retroviruses 7:645, 1991 ).
  • Immunomodulatory factors may be active both in vivo and ex vivo. Representative examples of such immunomodulatory factors include, for example, cytokines, such as IL-l, IL-2 (Karupiah et al, J. Immunology 144:290, 1990; Weber et al, J. Exp. Med.
  • immunomodulatory factor(s) include a-interferon, g-interferon, and IL-2 (see WO 94/21794).
  • Nucleic acid molecules that encode the above-described products, as well as other nucleic acid molecules that are advantageous for use within the present invention may be readily obtained from a variety of sources, including, for example, depositories such as the American Type Culture Collection, or from commercial sources such as British Bio- Technology Limited (Cowley, Oxford England). Alternatively, cDNA sequences for use with the present invention may be obtained from cells which express or contain the sequences, such as by RT PCR from isolated mRNA. Nucleic acid molecules suitable for use with the present invention may also be synthesized in whole or in part, for example, on an Applied Biosystems Inc. DNA synthesizer (e.g., ABl DNA synthesizer model 392 (Foster City, CA).
  • DNA synthesizer e.g., ABl DNA synthesizer model 392 (Foster City, CA).
  • a targeting element is a molecule that has affinity for a molecule present on the surface of a target cell.
  • targeting elements are considered to be "capable of interacting with a molecule present on the surface" of a selected cell type when a biological effect ofthe coupled targeting element may be seen in that cell type, or, when there is greater than at least about a 10-fold difference, and preferably greater than at least about a 25, 50, or 100-fold difference, between the binding ofthe targeting element to target cells and non-target cells.
  • the targeting element interact with a molecule present on the surface of the selected cell type with a KD of less than about 10" ⁇ M, preferably less than about 10 ⁇ 6M, more preferably less than about 10" ⁇ M, and most preferably less than about 10' ⁇ M (as determined by a Scatchard analysis, see Scatchard, Ann. N Y. Acad. Sci. 51:660, 1949).
  • Suitable targeting elements are preferably non-immunogenic, not degraded by proteolysis, and not scavenged by the immune system. Particularly preferred targeting elements should have a half-life within an animal of between about 10 minutes and about 1 week.
  • Suitable targeting elements include receptors, ligands, and antibodies (or antigen binding domains thereof).
  • Preferable receptor targeting elements include, for example, CD4 to target B-cells, CD8 to target T-cells, and CD21 to target B- cells.
  • Many other suitable receptors which can be used as targeting elements in accordance with the teachings provided herein are known in the art.
  • the targeting element is a ligand
  • it is preferably selected from a cytokine, lymphokine, polypeptide hormone, polypeptide or nonprotein molecule, for example, a carbohydrate.
  • Preferred cytokine ligands include IL-l type II to target myeloid cells or to target the interleukin- 1 receptor on T-cells (Fanslow et al, Science 245:739, 1990), IL2b to target B and T lymphocytes and monocytes, IL-3, SCF, or the flk-2 ligand to target hematopoietic cells, IL-6 to target activated B-cells, IL-7 to target lymphoid and myeloid cells, IL-8 to target T-cells and keratinocytes, and IL-10 to target mast cells.
  • Preferred lymphokine ligands include for example, GM-CSF to target granulocyte and monocyte lineage cells, G-CSF to target granulocyte lineage cells
  • Preferred polypeptide hormones include for example, follicle stimulating hormone (FSH) to target ovaries and testes, human growth hormone (HGH) to target osteocytes and myocytes, lutenizing hormone to target ovaries and testes, melanocyte stimulating hormone to target melanocytes, erythropoietin to target bone marrow cells, nerve growth factor to target nerve growth factor receptors on neural tumors (Chao et al, Science 232:518, 1986), vasoendothelial growth factor (VEGF) to target cells where increased vascularization occurs, and epidermal growth factor to target epidermal cells.
  • FSH follicle stimulating hormone
  • HGH human growth hormone
  • lutenizing hormone to target ovaries and testes
  • melanocyte stimulating hormone to target melanocytes
  • erythropoietin to target bone marrow cells
  • nerve growth factor to target nerve growth factor receptors on neural tumors
  • VEGF vasoendotheli
  • Preferred polypeptides include, for example, fimbriae to target CEA receptors on cancer cells, neuromedin (Conlon, J. Neurochem. 57:988, 1988) to target the cells ofthe uterus for contractile activity, insulin to target insulin receptors on cells for glucose regulation, the Fc receptor to target macrophages (Anderson and Looney, Immun. Today 7:264, 1987), transferrin to target transferrin receptors on tumor cells (Huebers et al, Physio. Rev.
  • asialoglycoprotein to target hepatocytes urokinase plasminogen activator (UP A) to target endothelial cells, lectins to target specific glycoproteins or glycolipids on the surface of target cells (Sharon and Lis, Science 246:227, 1989), collagen type I to target colon cancer (Pullam and Bodmer, Nature 356:529, 1992) and acetylated low density lipoproteins ("LDL”) to target macrophage scavenger receptors and atherosclerotic plaques (see Brown et al. , Ann. Rev.
  • LDL acetylated low density lipoproteins
  • a polypeptide targeting element which has affinity for a receptor on the target cell may be selected from libraries created utilizing recombinant techniques (see Scott and Smith, Science 249:386, 1990; Devlin et al, Science 249:404, 1990; Houghten et al, Nature 354:84 1991 ; Matthews and Wells, Science 260: ⁇ 1 13,1993 and Nissim et al, EMBO J. 73(3):692, 1994).
  • Preferred nonprotein molecules include for example, targeting elements selected from existing or created organic compound libraries. As stated above the targeting element may also be an antibody directed against a surface molecule ofthe target cell.
  • Preferred antibodies include 12.8 (Andrews et al, Blood 67:842, 1986), and MylO (Civin et ⁇ /., J. Immunol 733:157, 1984; commercially available from Becton Dickinson under the designation HPCA-2) to target the anti-CD34 antigen on stem cells, anti-CD4 antibody to target CD4+ T-cells, anti-CD8 antibodies to target CD8+ cells, the HER2/neu monoclonal antibody 4D5 (Sarup et al.
  • antibodies are preferably humanized. Techniques for the production of antibodies useful in the practice of this invention are known in the art.
  • targeting elements may be utilized that are capable of interacting with a molecule present on the surface of a selected cell type or when there is a greater than at least about 10-fold difference between the binding ofthe targeting element to the target cells and non-target cells.
  • Multifunctional linking agents are molecules that contain at least two reactive groups separated by a spacer or "bridge.”
  • multifunctional linking agents are used to covalently bind a targeting element to a GDV.
  • the spacer provides the spatial distance necessary to accommodate steric considerations ofthe moieties to be linked.
  • Different linking agents may be selected based on the lengths of bridges desired for the coupling.
  • a linking agent with a short spacer (4-8 A) is used and the degree of linking between the GDV and the targeting element is determined. If linking is minimal or unsuccessful, a multifunctional linking agent with a longer spacer is then selected. This process may be repeated in an iterative pattern until a linking agent providing the spacing is identified.
  • a bifunctional linking agent that has identical reactive groups on either end ofthe bridge is said to be homobifunctional. Where the reactive groups are different, the bifunctional linking agent is referred to as a heterobifunctional.
  • reactive groups include imidoesters, N-hydroxysuccinimidyl (NHS) esters, maleimides, pyridyl disulfides, carbodiimides, and arylazides, as well as others known in the art.
  • the imidoesters and the NHS esters react with primary amines present on the GDV and targeting element, while maleimide and pyridyl disulfide react with sulfhydryl groups present on the GDV and targeting element.
  • Carbodiimides couple carboxyl groups to primary amines present on the GDV and the targeting element.
  • An arylazide is a photoactivatable group that forms reactive nitrene when exposed to ultraviolet or visible light at wavelengths ranging from 250-460 nm. The aryl nitrene thus formed reacts nonselectively to form a covalent bond.
  • the targeting element is covalently bound to the GDV utilizing "multifunctional linking agents", preferably bifunctional linking agents (e.g., homobifunctional or heterobifunctional linking agents).
  • multifunctional linking agents e.g., homobifunctional or heterobifunctional linking agents.
  • a variety of multifunctional linking agents may be utilized and are available through Pierce (Rockford, IL).
  • Multifunctional linking agents include 4(4-N-maleimidophenyl)butyric acid hydride » HCl'l/2 dioxane (MPBH; a heterobifunctional non-cleavable linking agent), succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate (SMCC; a heterobifunctional linker), sulfosuccinimidyl (4(azidosalicylamido)hexanoate (sulfo-NHS- LC-ASA; a photoactivatable linking agent), sulfosuccinimidyl-2-[6-(biotinamido)-2-(p- azidobenzamido)-hexanoamido]ethyl-l,3'-dithiopropionate (Sulfo-SBED; a trifunctional linking agent having biotin covalently attached to a heterobifunctional reagent comprising a hydroxy
  • Multifunctional linking agents include, for example, N-succinimidyl-3- (2-pyridyl dithio) propionate (“SPDP”; Carlson et al, J. Biochem. 773:723, 1978), Sulfosuccinimidyl 4-N-maleimidomethyl) cyclohexane- 1 -carboxylate (“SulfoSMCC”), 1- ethyl-3 (3-dimethylaminopropyl) carbodiimide (“EDC”), and Bis-diazobenzidine (“BDB”).
  • SPDP N-succinimidyl-3- (2-pyridyl dithio) propionate
  • SulfoSMCC Sulfosuccinimidyl 4-N-maleimidomethyl) cyclohexane- 1 -carboxylate
  • EDC 1- ethyl-3 (3-dimethylaminopropyl) carbodiimide
  • BDB Bis-diazobenz
  • the multifunctional linking agent further comprises a monosaccharide, disaccharide or an oligosaccharide wherein the carbohydrate is first covalently bound to a targeting element utilizing the linking agents described above.
  • the modified targeting element is then covalently bound to a GDV via the carbohydrate moiety.
  • a targeting element is bound to a aminated carbohydrate utilizing a multifunctional linking agent.
  • a homobifunctional linker such as DSS may be utilized to covalently bind the targeting element to the carbohydrate via amine groups present on the targeting element and the aminated carbohydrate.
  • a heterobifunctional linker such as SMCC can be used to bind the carbohydrate to a sulfhydryl present on the targeting element to the amine group ofthe aminated carbohydrate.
  • the modified targeting element may then be bound to the GDV. Briefly, the GDV and the modified targeting element are mixed at various pHs ranging from about 7.4 to about 8.4 and incubated, preferably overnight at about 4°C. Following incubation, the mixture is treated with sodium cyanoborohydride. The reaction mixture is dialyzed at low temperature (about 2°C to 10°C) for a sufficient time (about 1 to 48 hours) to remove cyanoborohydride and sterilized by passage through an appropriate filter. Alternative procedures may be employed, depending on the carbohydrate and linker employed, as those in the art will appreciate.
  • GDV GDV Production
  • the GDV Once the GDV has been designed, it must be produced in an amount sufficient for conjugation to a desired targeting element and for administration to an animal.
  • the GDV is a recombinant viral vector, it may be produced utilizing a packaging system.
  • a variety of viral vector packaging systems are described below in which one or more essential functions ofthe parent virus has been deleted so that it is deficient in some function (e.g., genome replication), but retains a packaging signal and the ability to express gene products from one or more nucleic acid molecules.
  • Representative examples of viral vector packaging systems include those for retroviral vectors, alphaviral vectors and adenoviral vectors.
  • the deleted essential function or functions are provided by packaging cells into which the vector genome can be introduced to yield producer cell lines that then make viral particles encapsidating the recombinant viral vector.
  • such producer cell lines produce viral vectors substantially free from contamination with replication competent virus.
  • the vector genome is then introduced into target cells by an infection event ("transduction") but is incapable of further propagation. In any such situation, it is important to prevent the recombination ofthe various parts of the virus in a producer cell line to give replication competent virus genomes, or to eliminate cells in which this occurs.
  • the expression vector may be readily assembled from any virus utilizing standard recombinant techniques (e.g., Sambrook et. al, Molecular Cloning: A Laboratory Manual, 2d ed. Cold Spring Harbor Laboratory Press, 1989). Further description ofthe construction of retroviral vectors is described in WO 89/09271, herein inco ⁇ orated by reference.
  • the GDVs are retroviral vectors.
  • such vectors comprise a 5' LTR, a tRNA binding site, a packaging signal, one or more genes of interest, an origin of second strand DNA synthesis, and a 3' LTR, wherein the vector lacks gag/pol or env coding sequences.
  • a 5' LTR should be understood to include a 5' promoter element and sufficient LTR sequence to allow reverse transcription and integration ofthe DNA form ofthe vector.
  • the 3' LTR includes a polyadenylation signal and sufficient LTR sequence to allow reverse transcription and integration ofthe DNA form of the vector.
  • packaging cell lines for producing viral particles wherein at least the codons of 5' terminal end ofthe gag/pol gene are modified to take advantage ofthe degenerate nature ofthe genetic code to minimize the possibility of homologous recombination between the vector and sequences in the packaging cell coding for the viral structural proteins. Additional techniques for reducing the possibility of recombination events between vectors present in a packaging cell and the recombinant retroviral genome to be packaged are provided in WO 92/05266, WO 91/02805 and WO 96/20414.
  • Packaging cell lines suitable for use with the above described recombinant retroviral vectors may be readily prepared using techniques known in the art, and utilized to create producer cell lines for the production of recombinant vector particles.
  • alphavirus packaging cell lines are provided.
  • alphavirus packaging cell lines are provided wherein the viral structural proteins, supplied in trans from one or more stably integrated expression vectors, are able to encapsidate transfected, transduced, or intracellularly produced vector RNA transcripts in the cytoplasm and release infectious, packaged vector particles through the cell membrane, thus creating an alphavirus vector producing cell line.
  • Alphavirus RNA vector molecules capable of replicating in the cytoplasm ofthe packaging cell, can be produced initially utilizing, for example, an SP6 or T7 RNA polymerase system to transcribe in vitro a cDNA vector clone encoding the recombinant alphaviral genome which comprises the gene(s) of interest and the alphavirus non-structural proteins.
  • Vector RNA transcripts are then transfected into the alphavirus packaging cell line such that the vector RNA replicates to high levels and is subsequently packaged by viral structural proteins, yielding infectious vector particles.
  • Packaging cell lines suitable for use with the above described alphaviral vector constructs may be readily prepared (see WO 95/07994).
  • adenovirus packaging cell lines are provided.
  • Adenovirus vectors are derived from nuclear replicating viruses and may be constructed such that they are replication defective.
  • One or more nucleic acid molecules may be carried by adenoviral vectors for delivery to target cells (see Ballay et al. , EMBO J. 4:3861, 1985, Thummel et al, J. Mol. App Genetics 7:435, 1982 and WO 92/05266).
  • a targeted gene delivery vehicle may include one or more fusigenic proteins to assist in gene delivery.
  • Representative fusagenic proteins include ecotropic murine retrovirus envelope proteins, other retrovirus envelope proteins modified to disable normal receptor recognition, fusagenic proteins from he ⁇ es simplex virus fusagenic proteins gH and gL, Epstein-Barr virus fusagenic proteins, measles virus fusagenic proteins, malarial sporozoite fusagenic proteins, and other proteins known in the art to have fusogenic properties.
  • compositions comprising targeted GDVs are preferably purified again prior to administration.
  • the techniques utilized for purification is dependent on the type of GDV to be purified. For example, there are a variety of techniques known in the art which may be used if the GDV is an enveloped recombinant viral vector, a nucleic acid or a liposome. A preferred method is described in co-owned U.S. Patent No. 5,447,859, issued September 5, 1995.
  • the GDVs are typically purified to a level ranging from 0.25% to 25%, and preferably about 5% to 20% before conjugation.
  • the GDV is a nucleic acid
  • there are a variety of techniques known in the art including, for example, purification by CsCl-ethidium bromide gradient, ion- exchange chromatography, gel-filtration chromatography, and differential precipitation with polyethylene glycol. Further description ofthe purification of nucleic acids is provided in Sambrook et. al, Molecular Cloning: A Laboratory Manual, 2d ed. (Cold Spring Harbor Laboratory Press, 1989).
  • preparation of liposomes typically involves admixing solutions of one or more purified phospholipids and cholesterol in organic solvents and evaporating the solvents to dryness. An aqueous buffer containing the GDVs is then added to the lipid film and the mixture is sonicated to create a fairly uniform dispersion of liposomes. In certain embodiments, dialysis, gel filtration, or ultracentrifugation is then be used to separate uninco ⁇ orated components from the intact liposomes.
  • linking agents may be utilized to bind target elements to GDV.
  • the methods used vary depending on the available functional groups on the exterior ofthe GDV and the targeting element.
  • a multifunctional linking agent such as disuccinimidyl suberate (DSS, Pierce, Rockford, IL) which is a homobifunctional N- hydroxysuccinimdyl ester linking agent.
  • DSS disuccinimidyl suberate
  • IL a homobifunctional N- hydroxysuccinimdyl ester linking agent
  • the GDV contains sulfhydryl functional groups on its surface and the targeting element has a primary amine available a heterobifunctional linking agent may be utilized for example succinimidyl 4-(N- maleimidomethyl)cyclohexane-l -carboxylate.
  • GDV contains a carboxyl functional group and the targeting element has a sulfhydryl functional group then a heterobifunctional group such as 4(4-N-maleimidophenyI)butyric acid hydride*HCM/2 dioxane may be utilized.
  • Other linking agents may be utilized include trifunctional linking agents which permit binding of a targeting element, a GDV and a third element and light activated linking agents see Example 4 below.
  • GDV can be bound to targeting elements by a variety of other methods.
  • GDV/targeting element similar to a cesium chloride gradient (see Sambrook et al, supra).
  • the preparation is preferably formulated into a pharmaceutical composition containing some or all ofthe following: one or more pharmaceutically acceptable carriers and/or diluents; a saccharide; a high molecular weight structural additive; a buffering component; water; and one or more amino acids.
  • a pharmaceutically acceptable carriers or diluents are non-toxic to recipients at the dosages and concentrations employed.
  • carriers or diluents for injectable solutions include for example water, isotonic saline solutions (i.e., phosphate- buffered saline or Tris-buffered saline, preferably buffered at physiological pH), mannitol, dextrose, glycerol, and ethanol, as well as polypeptides or proteins such as human serum albumin.
  • the saccharide provides, among other things, support in the lyophilized or dried state.
  • the preferred saccharide is lactose
  • other saccharides may be used, such as sucrose, mannitol, glucose, trehalose, inositol, fructose, maltose or galactose.
  • combinations of saccharides can be used, for example, lactose and mannitol, or sucrose and mannitol.
  • a particularly preferred concentration of lactose is 3% to 4% by weight.
  • the concentration ofthe saccharide ranges from 1% to 12% by weight.
  • a preferred composition comprises 10 mg/mL mannitol, 1 mg/mL HSA, 20 mM Tris, pH 7.2, and 150 mM NaCl being particularly preferred (see WO 95/10601). Such compositions are stable at -70°C for at least six months.
  • compositions ofthe invention may also additionally include factors to stimulate cell division, and hence, uptake and inco ⁇ oration ofthe administered
  • GDVs include melanocyte stimulating hormone (MSH) for melanoma, epidermal growth factor (EGF) for breast or other epithelial carcinomas, and the anesthetic bipuvocaine (or related compounds) for intramuscular injection.
  • MSH melanocyte stimulating hormone
  • EGF epidermal growth factor
  • bipuvocaine bipuvocaine
  • differentially targeting GDVs i.e., GDVs targeted to different tissues, or the same tissue by way of a different interaction
  • GDVs targeted to different tissues may be provided in a single composition or each targeted GDV may be administered separately to an animal.
  • compositions containing multiple targeted GDVs are typically administered in the same composition, but may be simultaneously administered at the same time and same site, such as via the use of a double barreled syringe or by joint formulation.
  • a composition containing one or more different targeted GDVs may also be administered at different sites, as disclosed in WO 96/20731.
  • compositions according to the invention may be provided either as a liquid solution, or as a solid form (e.g., lyophilized or dehydrated) which can be resuspended in a solution prior to administration.
  • lyophilization involves the steps of cooling the aqueous suspension below the glass transition temperature or below the eutectic point temperature ofthe aqueous suspension, and removing water from the cooled suspension by sublimation. See Phillips et al, Cryobiology 75:414, 1981 and WO 95/10601.
  • the resulting composition preferably contains less than 10% water by weight.
  • the composition is stable and may be stored at or above -70°C preferably at - 20°C to -25°C.
  • water is removed from the aqueous suspension at ambient temperature by evaporation.
  • water may be removed through spray drying (see EPO 520,748).
  • Spray drying apparatus are available from a number of manufacturers (e.g., Drytec, Ltd., Tonbridge, England; Lab-Plant, Ltd., Huddersield, England).
  • the targeted GDV composition is stable and may be stored at or above -70°C preferably at -20 C to -25 C.
  • the recombinant virus will constitute about 10 ng to 1 mg of material per dose, with about 10 times this amount of material present as copurified contaminants.
  • the composition is administered in doses of about 0.1 to 1.0 mL of aqueous solution, which may or may not contain one or more additional pharmaceutically acceptable excipients, stabilizers, or diluents.
  • compositions are typically administered in vivo via traditional direct routes, such as buccal/sublingual, rectal, oral, nasal, topical, (such as transdermal and ophthalmic), vaginal, pulmonary, intraarterial, intramuscular, intraperitoneal, subcutaneous, intraocular, intranasal, intravenous routes or directly into a specific tissue, such as the liver, bone marrow, or into the tumor in the case of cancer therapy.
  • direct routes such as buccal/sublingual, rectal, oral, nasal, topical, (such as transdermal and ophthalmic), vaginal, pulmonary, intraarterial, intramuscular, intraperitoneal, subcutaneous, intraocular, intranasal, intravenous routes or directly into a specific tissue, such as the liver, bone marrow, or into the tumor in the case of cancer therapy.
  • the composition is administered to an animal via the desired route and then the animal is tested for the desired biological response.
  • testing may include immunological screening assays e.g., CTL assays, antibody assays.
  • the test(s) performed will depend on the product produced by the nucleic acid molecule introduced by the targeted GDV and the disease to be treated or prevented.
  • the titers ofthe targeted GDVs to be administered may be adjusted to further enhance the desired effect(s).
  • the following examples are offered by way of illustration and not by way of limitation.
  • Linking agents are utilized to link GDVs to the selected targeting elements. These linking agents contain at least two reactive groups which, upon activation in the presence of GDV and the targeting element, form covalent bonds thereby coupling the GDV and targeting element. When the reactive groups are identical, the linking agent is said to be homobifunctional. If the reactive groups are different, the linking agent is referred to as a heterobifunctional. Examples of reactive groups include imidoesters, N- hydroxysuccinimidyl (NHS) esters, maleimides, pyridyl disulfides, carbodiimides, and arylazides.
  • reactive groups include imidoesters, N- hydroxysuccinimidyl (NHS) esters, maleimides, pyridyl disulfides, carbodiimides, and arylazides.
  • the imidoesters and the NHS esters react with primary amines present on GDVs and targeting elements while the maleimide and pyridyl dissulfide react with sulfhydryl groups present on GDVs and targeting elements.
  • Carbodiimides couple carboxyl groups to primary amines present on the GDV and the targeting elements.
  • a photoactivatable arylazide is a group that is photolyzed when exposed to ultraviolet or visible light at wavelengths ranging from 250-460 nm to form a reactive nitrene. The aryl nitrene thus formed non-selectively forms a covalent bond.
  • the linking agent may further comprise a monosaccharide, disaccharide or an oligosaccharide, wherein the carbohydrate is covalently bound to a targeting element utilizing the linking agents described above and then the modified targeting element is covalently bound to a GDV via the carbohydrate.
  • HCM/2 dioxane 4(4-N-maleimidophenyl)butyric acid hydride » HCM/2 dioxane (MPBH, Pierce, Rockford, IL) is a heterobifunctional non-cleavable linking agent containing a hydrazide group and maleimide that react with carbohydrates and sulfhydryls, respectively.
  • This protocol provides for the conjugation of glycoproteins present on the surface of a recombinant virus, a polycation, a liposome, bacteriophage or bacterium to thiol-containing proteins of a targeting element.
  • the recombinant virus is first conjugated to MPBH followed by conjugating to the sulfhydryl-containing targeting element.
  • reaction mixture is brought back to its original volume with 0.1 M sodium acetate buffer, pH 5.5, and the centrifugation procedure is repeated two additional times.
  • a 10 mg/mL solution of MPBH is added to the oxidized recombinant virus to a final concentration of 1 mM MPBH and allowed to react with agitation for 2.0 hours at room temperature.
  • the excess MPBH is removed by centrifugation at 1000 x g for 15 to 30 minutes using a microconcentrator.
  • the sample is then brought back to its original volume in 0.1 M sodium phosphate, 50 mM NaCl, pH 7.0. This centrifugation process is repeated twice.
  • the targeting element 5 mg/mL ofthe targeting element in 0.1 M sodium phosphate, 50 mM NaCl, pH 7.0 buffer
  • This reaction mixture is incubated for 2.0 hours at room temperature.
  • the targeting element-conjugated recombinant virus may then be purified by column chromatography.
  • Succinimidyl 4-(N-maleimidomethylteyclohexane-l -carboxylate (Pierce, Rockford, IL) linking agent consists of an N-hydroxysuccinimide (NHS)-ester and a maleimide group connected with a spacer.
  • NHS ester reacts with a primary amine at pH 7 to 9 and the maleimide reacts with sulfhydryl groups at pH 6.5 to 7.5.
  • This protocol provides for the conjugation of a recombinant virus, a polycation, a liposome, bacteriophage or bacterium to a targeting element via sulfhydryls present in the viral coat proteins ofthe recombinant virus to the primary amines present on the targeting element.
  • a GDV solution is prepared to an equivalent protein concentration of 4.0 mg/mL in a phosphate buffered saline (PBS) solution containing EDTA to a final concentration of 5 mM.
  • PBS phosphate buffered saline
  • a reducing agent is added to this solution containing 0.5 M b-2-mercaptoethanolamine in PBS with 5.0 mM EDTA and the reaction mixture is incubated for 90 minutes at 37°C. Following incubation the modified GDV may be desalted by column chromatography.
  • the maleimide activated-targeting element may be desalted by column chromatography.
  • the reduced GDV is mixed with the maleimide activated-targeting element and incubated at 4°C overnight.
  • the targeting element conjugated GDV may be desalted by column chromatography.
  • the reaction mixture is incubated for 15 minutes at 37°C and then irradiated with long wave UV light for 10 minutes at room temperature. This mixture is then flashed with a bright light for 1 to 3 seconds (three camera flashes).
  • the photoactivated targeting element conjugated GDV may be desalted by column chromatography.
  • Sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl- 1 ,3'-dithiopropionate is a trifunctional crosslinking reagent having biotin covalently attached to a heterobifunctional reagent comprising a hydroxysuccinimido active ester and a photoreactive aryl azide. Approximately 1.12 mg of Sulfo-SBED is dissolved in 25 ⁇ L DMSO.
  • the Sulfo-SBED is then added to a solution of targeting element containing 5 mg ofthe targeting element in 0.5 mL of 0.1 M PBS, pH 7.2. This mixture is incubated at room temperature for 30 minutes.
  • the linking agent-targeting element conjugate may be desalted by column chromatography.
  • the linking agent-targeting element conjugate is then mixed with the GDV solution having an equivalent protein concentration of 5.0 mg dissolved in 0.5 mL PBS and incubated at room temperature for 3.5 minutes. This reaction mixture is irradiated with long wave UV light for 15 minutes.
  • the targeting element-GDV conjugate may be desalted by column chromatography. Since the linking agent is biotinylated a second molecule conjugated to avidin may be bound to this targeting element-GDV conjugate.
  • Disuccinimidyl suberate (DSS, Pierce, Rockford, IL) is a homobifunctional N- hydroxysuccinimdyl ester linking agent.
  • This protocol provides for the conjugation ofa GDV to a targeting element via primary amines present on the proteins ofthe GDV and the targeting element.
  • a protein concentration equivalent of 0.1 to 0.5 mg ofthe GDV in PBS is incubated with the targeting element having a concentration of 5 to 10 nM in PBS in a total volume of 100 ⁇ L for 1.0 hour at 4°C.
  • DSS solution DSS dissolved in dry DMSO to a 10-25 mM concentration
  • a stop solution 1.0 M Tris, pH 7.5
  • the targeting element-GDV may be desalted by column chromatography.
  • the MSH-glucose conjugate is added to a cold solution of sodium wet ⁇ -periodate containing 100 mM sodium periodate in 0.1 M sodium acetate buffer, pH 5.5.
  • the oxidation reaction is allowed to proceed for 1.0 hour in the dark at room temperature.
  • Glycerol is added and the mixture is dialyzed at 4°C overnight and then concentrated.
  • a liposomal suspension (1.5 ⁇ mole) is mixed with l-ethyl-3-(3- dimethylaminopropyl)-carbodiimide (EDC; 4 mg, Pierce Chemical Co., Rockford, IL) in 1.5 mL of 10 mM NaPO4, 0.15 M NaCl, pH 5.0. The reaction is carried out at room temperature for one hour.
  • EDC l-ethyl-3-(3- dimethylaminopropyl)-carbodiimide
  • the liposome/EDC mixture (1.5 mL) is mixed with 75 ⁇ L of mouse IgG (Cappel
  • the amount of protein bound to the liposome is determined by the Lowry protein assay.
  • the concentration of lipid is determined from I '25 radioactivity levels, based on a known amount of PE-I ⁇ 5 included in the liposome preparations. Based on the measured protein and lipid concentrations, the protein to lipid coupling ratios, expressed in micrograms protein/ ⁇ mole, lipid concentrations are determined.
  • HepG2 Human hepatoma cell lines HepG2 (Schwartz, et al , J. Biol. Chem. 256:8878, 1981 ) and SK Hep 1, and rat hepatoma cell line Morris 7777 (ATCC CRL 1601, Wu et. al, J. Biol. Chem. 263:4719, 1988) and murine fibroblast cell line NIH3T3 (ATCC CRL 1658,
  • Goud et al, Vir. 763:251, 1988) are plated at a density of 0.5 to 2.0 x 10 5 cells/mL in 60 mm plastic dishes (Falcon Scientific Co., Lincoln Park, NJ).
  • Equal amounts (16.7 ⁇ g of RNA, 0.5 mg of viral protein) of modified and unmodified GDV in Dulbecco's modified Eagle's medium are added to the culture medium and exposed to cells for 5 days at 37°C under 5% CO2- Cells are assayed for b-galactosidase activity as a measure of foreign gene expression according to the method of Gorman (DNA Cloning 2:157-158, 1986, Glover, D.M., ed., IRL Press, Washington, DC).
  • cell monolayers (approximately 1.0 x 10 ⁇ cells/60 mm dish) are washed with phosphate-buffered saline, then lysed.
  • the lysate 0.1 mL, is reacted with o-nitrophenyl galactopyranoside (Sigma, St. Louis, MO) and b- galactosidase activity quantitated by absorbance at 420 nm after the addition of Na2CO3 to terminate the reaction.
  • phosphate-buffered saline then incubated with 1.0 mM MgCl2 phosphate-buffered saline, and overlaid with 1.0 mg/mL X-gal (GIBCO, Bethesda Research Laboratories, MD), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 2 mM MgCl2 in phosphate-buffered saline. After incubation at 37°C for 1 hour, the dishes are washed in phosphate-buffered saline to quench the reaction and evaluated by counting positive (blue) cells under a light microscope, and the results are expressed as the percent of positive 10 high power fields.
  • the cells may be incubated at 37°C in serum-free Dulbecco's modified Eagle's medium containing 3 s-biolabeled, modified GDV, 3.3 ⁇ g of viral RNA (98 ⁇ g of viral protein) (Watanabe, et al, Cancer Immunol. Immunother. 25:157, 1989) with a specific activity of 6.1 x 10 ⁇ cpm/mg of nucleic acid.
  • medium is removed, and cells are chilled to 4°C and washed with ice-cold minimum essential medium containing 1.0 mg/mL bovine serum albumin.
  • Radioactivity is stripped with 0.5 mL of cold phosphate-buffered saline, pH 7.2, containing 0.4% trypsin, 0.02% EDTA, and separated from cells by centrifugation.
  • the cell pellet is solubilized in 0.2 N NaOH and Poly-Fluor (Packard Instrument Co.), and trypsin-EDTA-resistant (internalized) radioactivity is measured by scintillation counting (Tr-Carb 4530, Packard) (Schwartz, et al, J. Biol. Chem. 256:8878, 1981).
  • Nonspecific uptake is measured in the presence of a 100-fold molar excess of targeting element, and specific uptake is calculated as the difference between total and nonspecific measurements.
  • samples of freshly prepared sterile, GDV conjugated to the desired targeting element are incubated in serum-free Dulbecco's modified Eagle's medium at 4 and 25°C. At various times, samples are added to the medium of target cells and incubated for 5 days. Cells are then assayed for b-galactosidase activity by colorimetric assay as described above.
  • Core antigen and precore antigen in cell lysates and secreted e antigen in culture supernatant are assayed using the Abbott HBe, rDNA EIA kit (Abbott Laboratories Diagnostic Division, Chicago, IL).
  • Another sensitive EIA assay for precore antigen in cell lysates and secreted e antigen in culture supernatant is performed using the Incstar ETI-EB kit, (Incstar Co ⁇ oration, Stillwater, MN).
  • a standard curve is generated from dilutions of recombinant hepatitis B core and e antigen obtained from Biogen (Geneva, Switzerland).
  • HBV e antigen approximately 20-40 ng/ml HBV e antigen is expressed in transduced cell lines, and 38-750 ng/ml of HBV core antigen is expressed in transduced cell lines.
  • protein expression may be determined by Western blot or by Immunoprecipitation Western blot. See U.S.S.N. 08/483,51 1, filed June 7, 1995.
  • expression may be assayed by exposing the sample to luciferin and measuring the resulting luminescence. Briefly, transfected cells are harvested, washed in PBS and resuspended in 200 mL of 0.25 M Tris- HCl, pH 7.8. The cells are lysed by three cycles of freeze/thawing and the cellular debris is removed by centrifugation.
  • luciferase activity Approximately 50 mL of cell lysate is assayed for luciferase activity by measuring light emission with a bioluminometer (Analytical Bioluminescence, San Diego, CA) in the presence of luciferin and ATP (Brasier et al, Biotechniques 7:11 16, 1989). The amount of protein in the lysate is determined by the Bradford dye-binding procedure (Bio-Rad, Hercules, CA).
  • Targeted GDV preparations made in accordance with the teachings provided herein are injected into an animal at doses of 10 5 , IO 6 , IO 7 , 10 8 , IO 9 , IO 10 , or 10 1 ] GDVs with or without uptake enhancers such as polybrene (1-8 ⁇ g/mL) or DEAE dextran (2 - 30 ⁇ g/mL).
  • uptake enhancers such as polybrene (1-8 ⁇ g/mL) or DEAE dextran (2 - 30 ⁇ g/mL).
  • Injections are given daily for 1, 2, 3, 4, 5, 6, or 7 days, and 2 to 7 days after the last injection, to determine the activity ofthe delivered gene. Injections are typically administered through an I.V.
  • Patients preferably receive doses of about 10 6 , IO 7 , IO 8 , IO 9 , 10 10 , or 10* l targeted GDVs I.V., intra-arterially, in the local vasculature or peritumorally, as the case may be, in a volume of 0.1 to 3 mL.
  • the gene is one that encodes a protein which converts a non-toxic precursor (prodrug) into a toxic product
  • the prodrug is administered at doses defined in the Physicians Desk Reference or those predicted from animal experiments at times of between 1 to 30 days after the last administration of the targeted GDV.
  • the targeted GDV is typically administered from 1 to 20 times al intervals of 1 to 15 days and the patient status is monitored by following normal clinical parameters and monitoring tumor sizes by radiography, MRl scans, PET scans or other conventional means.

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Abstract

Compositions et procédés permettant de doter des vecteurs d'acheminement de gènes d'éléments de ciblage. Spécifiquement, la présente invention concerne l'utilisation d'agents de liaison multifonctionnels, par ex. des agents de liaison homobifonctionnels, hétérobifonctionnels et trifonctionnels, pour lier de manière covalente des éléments de ciblage à l'extérieur de vecteurs d'acheminement de gènes. Suite à l'administration de ces vecteurs d'acheminement de gènes dotés d'éléments de ciblage à un animal, lesdits éléments interagissent avec un module spécifique sur la surface des cellules cibles, après quoi la molécule d'acide nucléique désirée est introduite dans la cellule cible et exprimée. Le mécanisme de ciblage décrit permet d'acheminer des vecteurs d'acheminement de gènes à des cellules ou tissus cibles spécifiques avec une spécificité plus grande que celle rencontrée lorsque le vecteur d'acheminement de gènes administré est exempt d'éléments de ciblage liés de manière covalente.
PCT/US1996/020543 1995-12-22 1996-12-18 Compositions et procedes permettant de doter des vecteurs d'acheminement de genes d'elements de ciblage lies de maniere covalente WO1997023608A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6060316A (en) * 1998-06-09 2000-05-09 President And Fellows Of Harvard College Methods of targeting of viral entry
WO2000037103A2 (fr) * 1998-12-21 2000-06-29 Xavos Composes d'apport intracellulaire de groupes caracteristiques therapeutiques a des cellules nerveuses
US6660264B1 (en) 1999-04-09 2003-12-09 Health Protection Agency Treatment of intracellular infection
US6866860B2 (en) 2002-12-19 2005-03-15 Ethicon, Inc. Cationic alkyd polyesters for medical applications
US8232251B2 (en) 1998-12-21 2012-07-31 Manzanita Pharmaceuticals, Inc. Compounds for delivery of therapeutic and imaging moieties to nerve cells
US11897928B2 (en) 2018-07-18 2024-02-13 Manzanita Pharmaceuticals, Inc. Conjugates for delivering an anti-cancer agent to nerve cells, methods of use and methods of making thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4115038A1 (de) * 1991-05-08 1992-11-12 Genentech Inc Neue konjugate, bestehend aus einem glykoprotein und einer nukleinsaeure-bindenden substanz
WO1994010323A1 (fr) * 1992-11-04 1994-05-11 Imperial Cancer Research Technology Limited Virus comprenant une fraction de liaison modifiee specifique contre des cellules cibles
WO1994025608A1 (fr) * 1993-04-27 1994-11-10 Baylor College Of Medicine Proteines de fixations d'adn naturel ou recombine utilisees comme vecteurs dans le transfert de genes ou la therapie genique
WO1995031566A1 (fr) * 1994-05-13 1995-11-23 Chiron Viagene, Inc. Compositions et procedes de ciblage de vecteurs d'apport de genes
WO1996015811A1 (fr) * 1994-11-17 1996-05-30 Imperial College Of Science, Technology & Medicine Internalisation de l'adn utilisant des conjugues de poly-l-lysine et d'un ligand se fixant aux recepteurs de l'integrine

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4115038A1 (de) * 1991-05-08 1992-11-12 Genentech Inc Neue konjugate, bestehend aus einem glykoprotein und einer nukleinsaeure-bindenden substanz
WO1994010323A1 (fr) * 1992-11-04 1994-05-11 Imperial Cancer Research Technology Limited Virus comprenant une fraction de liaison modifiee specifique contre des cellules cibles
WO1994025608A1 (fr) * 1993-04-27 1994-11-10 Baylor College Of Medicine Proteines de fixations d'adn naturel ou recombine utilisees comme vecteurs dans le transfert de genes ou la therapie genique
WO1995031566A1 (fr) * 1994-05-13 1995-11-23 Chiron Viagene, Inc. Compositions et procedes de ciblage de vecteurs d'apport de genes
WO1996015811A1 (fr) * 1994-11-17 1996-05-30 Imperial College Of Science, Technology & Medicine Internalisation de l'adn utilisant des conjugues de poly-l-lysine et d'un ligand se fixant aux recepteurs de l'integrine

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
C.H. WU ET AL.: "Targeting genes: delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in vivo", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 264, no. 29, 15 October 1989 (1989-10-15), US, pages 16985 - 16987, XP002030967 *
CURIEL D T ET AL: "ADENOVIRUS ENHANCEMENT OF TRANSFERRIN-POLYLYSINE-MEDIATED GENE DELIVERY", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 88, October 1991 (1991-10-01), pages 8850 - 8854, XP002001452 *
WAGNER E ET AL: "DNA-BINDING TRANSFERRIN CONJUGATES AS FUNCTIONAL GENE-DELIVERY AGENTS: SYNTHESIS BY LINKAGE OF POLYLYSINE OR ETHIDIUM HOMODIMER TO THE TRANSFERRIN CARBOHYDRATE MOIETY", BIOCONJUGATE CHEMISTRY, vol. 2, no. 4, 1 January 1991 (1991-01-01), pages 226 - 231, XP002002761 *
WAGNER E ET AL: "INFLUENZA VIRUS HEMAGGLUTININ HA-2 N-TERMINAL FUSOGENIC PEPTIDES AUGMENT GENE TRANSFER BY TRANSFERRIN-POLYLYSINE-DNA COMPLEXES: TOWARD A SYNTHETIC VIRUS-LIKE GENE-TRANSFER VEHICLE", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 89, no. 17, 1 September 1992 (1992-09-01), pages 7934 - 7938, XP000371760 *
WAGNER E ET AL: "TRANSFERRIN-POLYCATION CONJUGATED AS CARRIERS FOR DNA UPTAKE INTO CELLS", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 87, 1 May 1990 (1990-05-01), pages 3410 - 3414, XP002002759 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6060316A (en) * 1998-06-09 2000-05-09 President And Fellows Of Harvard College Methods of targeting of viral entry
US7718605B2 (en) 1998-12-21 2010-05-18 Manzanita Pharmaceuticals, Inc. Compounds for intracellular delivery of therapeutic moieties to nerve cells
WO2000037103A3 (fr) * 1998-12-21 2000-10-19 Xavos Composes d'apport intracellulaire de groupes caracteristiques therapeutiques a des cellules nerveuses
JP2002532560A (ja) * 1998-12-21 2002-10-02 ザヴォス 治療成分を神経細胞に細胞内送達するための化合物
US6652864B1 (en) 1998-12-21 2003-11-25 Asilomar Pharmaceuticals, Inc. Compounds for intracellular delivery of therapeutic moieties to nerve cells
US6887861B1 (en) 1998-12-21 2005-05-03 Asilomar Pharmaceuticals, Inc. Compounds for intracellular delivery of therapeutic moieties to nerve cells
US7678378B2 (en) 1998-12-21 2010-03-16 Manzanita Pharmaceuticals, Inc. Compounds for intracellular delivery of therapeutic moieties to nerve cells
WO2000037103A2 (fr) * 1998-12-21 2000-06-29 Xavos Composes d'apport intracellulaire de groupes caracteristiques therapeutiques a des cellules nerveuses
US8138155B2 (en) 1998-12-21 2012-03-20 Manzanita Pharmaceuticals, Inc. Compounds for intracellular delivery of therapeutic moieties to nerve cells
US8232251B2 (en) 1998-12-21 2012-07-31 Manzanita Pharmaceuticals, Inc. Compounds for delivery of therapeutic and imaging moieties to nerve cells
US6660264B1 (en) 1999-04-09 2003-12-09 Health Protection Agency Treatment of intracellular infection
US6866860B2 (en) 2002-12-19 2005-03-15 Ethicon, Inc. Cationic alkyd polyesters for medical applications
US11897928B2 (en) 2018-07-18 2024-02-13 Manzanita Pharmaceuticals, Inc. Conjugates for delivering an anti-cancer agent to nerve cells, methods of use and methods of making thereof

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