EP0693130A1 - Apport cible de genes codant des polyribonucleotides antisens - Google Patents

Apport cible de genes codant des polyribonucleotides antisens

Info

Publication number
EP0693130A1
EP0693130A1 EP94912389A EP94912389A EP0693130A1 EP 0693130 A1 EP0693130 A1 EP 0693130A1 EP 94912389 A EP94912389 A EP 94912389A EP 94912389 A EP94912389 A EP 94912389A EP 0693130 A1 EP0693130 A1 EP 0693130A1
Authority
EP
European Patent Office
Prior art keywords
gene
rna
cell
complex
soluble
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94912389A
Other languages
German (de)
English (en)
Inventor
Henry C. Chiou
Steven F. Innaimo
George Y. Wu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Connecticut
TargeTech Inc
Original Assignee
University of Connecticut
TargeTech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Connecticut, TargeTech Inc filed Critical University of Connecticut
Publication of EP0693130A1 publication Critical patent/EP0693130A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • 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
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide

Definitions

  • Antisense polynucleotides are a means of specifically inhibiting unwanted gene expression in cells. They can be used to hybridize to and inhibit the function of an RNA, typically a messenger RNA, by physically blocking the binding of ribosomes or other proteins, thus preventing translation of the mRNA. Antisense polynucleotides also include RNAs with catalytic activity (ribozymes), which can selectively bind to complementary sequences on a target RNA and physically destroy the target by mediating a cleavage reaction.
  • ribozymes RNAs with catalytic activity
  • Antisense polynucleotides can be in the form of small, chemically synthesized DNA or RNA oligonucleo- tides, or can be larger RNAs, such as mRNAs, biosynthetically generated in vitro or in vivo by transcription of an antisense gene. Since hundreds of copies of RNA can be synthesized from each copy of a gene, a few molecules of an antisense gene in a cell would achieve the same effect as the introduction into the cell of a large number of antisense oligonucleo- tides. The 50 to 300 times greater size of a typical mRNA allows an antisense mRNA to bind with greater affinity and specificity compared to an oligonucleo- tide.
  • a plasmid-borne gene can incorporate a wide variety of supplemental DNA sequences to enhance or modulate the expression of the antisense polyribonucleotide.
  • inclusion of origin sequences which direct episomal replication of a plasmid and promoter and/or enhancer sequences that sustain a hig level of expression over long periods would allow long term production of an antisense polyribonucleotide.
  • This strategy would be particularly suited for inhibiting the constitutive expression of a cellular gene or for treating chronic conditions or diseases.
  • To achieve a similar effect with an antisense oligonucleotide would most likely require continuous or repeated intravenous administration or highly stable antisense constructs.
  • This invention pertains to a soluble molecular complex for targeting a gene encoding an RNA transcript to a specific cell in vivo or in vitro and obtaining production of the RNA within the targeted cell.
  • the molecular complex comprises an expressible gene encoding a desired polyribonucleotide complexed to a carrier which is a conjugate of a cell-specific binding agent and a gene-binding agent.
  • the RNA transcribed from the delivered gene can be used to hybridize to and inhibit the function of an RNA contained within the cell.
  • the target RNA is typically a messenger RNA.
  • the RNA transcribed from the delivered gene can also be an RNA with catalytic activity (a ribozyme), which can selectively destroy the target RNA.
  • the target for antisense or ribozyme-mediated inhibition can be a gene or genes of cellular origin (e.g., a cellular oncogene) or of noncellular origin (e.g., a viral oncogene or the genes of an infecting pathogen such as a virus or a parasite such as malaria, trypanosome, lysteria, or mycoplasma).
  • cellular origin e.g., a cellular oncogene
  • noncellular origin e.g., a viral oncogene or the genes of an infecting pathogen such as a virus or a parasite such as malaria, trypanosome, lysteria, or mycoplasma.
  • the cell-specific binding agent is specific for a cellular surface structure, typically a receptor, which mediates internalization of bound ligands by endocytosis, such as the asialoglycoprotein receptor of hepatocytes.
  • the cell-specific binding agent can be a natural or synthetic ligand (for example, a protein, polypeptide, glycoprotein, etc.) or it can be an antibody, or an analogue thereof, which specifically binds a cellular surface structure which then mediates internalization of the bound complex.
  • the gene-binding component of the conjugate is a compound such as a polycation which stably complexes the gene under extracellular conditions and releases the gene under intracellular conditions so that it can function within the cell.
  • the complex of the gene and the carrier is stable and the carrier is stable and soluble in physiological fluids. It can be administered in vivo where it is selectively taken up by the target cell via the surface-structure-mediated endocytotic pathway. The incorporated gene expressed and the gene-encoded product accumulates within the transfected cell.
  • the soluble molecular complex of this invention can be used to specifically transfect cells in vivo or in vitro to provide for synthesis of a desired product. This selective transfection is useful for antisense gene therapy and other applications which require selective genetic alteration of cells to inhibit the expression of cellular or foreign genes.
  • the RNA transcript produced from the delivered gene hybridizes with its complementary RNA, inhibiting its function either by steric hindrance, or by physical cleavage, thereby blocking expression of the target gene or genes.
  • Figure 1 is a schematic depiction of the construction of a plasmid encoding an antisense RNA directed against hepatitis B surface antigen (pJ3 ⁇ 0.8HTDl) and a plasmid encoding an antisense RNA directed against the hepatitis core antigen gene as well as the
  • Figure 2 shows the reduction of HBsAg mediated by 21 -mer antisense oligodeoxynucleotide and antisense mRNA-generating plasmid pJ3 ⁇ 0.8HTDl (Anti-C).
  • a soluble, targetable molecular complex is used to selectively deliver a gene encoding a polyribonucleotide to a target cell or tissue in vivo or in vitro.
  • the molecular complex comprises the gene to be delivered complexed to a carrier made up of a binding agent specific for the target cell and a gene-binding agent specific for the target cell and a gene-binding agent.
  • the complex is selectively taken up by the target cell and the polyribonucleotide is produced therein.
  • the gene generally in the form of DNA, encodes the desired polyribonucleotide.
  • the gene comprises a sequence encoding the polyribonucleotide in a form suitabl for transcription and post-transcriptional processing by the target cell.
  • the ge is linked to appropriate genetic regulatory elements required for transcription of the gene b cellular RNA polymerase and processing of the primary RNA transcript by cellular protein into a stable form of RNA, such as mRNA.
  • RNA such as mRNA
  • promoter and enhancer elements operable in the target cell, as well as other elements such as polyadenylation signals and splicing signals, which determine the internal and 3 '-end structure of the RNA.
  • the gene c be contained in an expression vector such as a plasmid or a transposable genetic element along with the genetic regulatory elements necessary for transcription of the gene.
  • the carrier component of the complex is a conjugate of a cell-specific binding agent and a gene-binding agent.
  • the cell-specific binding agent specifically binds a cellular surfa structure which mediates its internalization by, for example, the process of endocytosis.
  • Th surface structure can be a protein, polypeptide, carbohydrate, lipid or combination thereof. is typically a surface receptor which mediates endocytosis of a ligand.
  • the binding agent can be a natural or synthetic ligand which binds the receptor.
  • the ligand can be a protein, polypeptide, glycoprotein or glycopeptide which has functional groups that are exposed sufficiently to be recognized by the cell surface structure. It can also be a component of a biological organism such as a virus, cells (e.g., mammalian, bacterial, protozoan) or artificial carriers such as liposomes.
  • the binding agent can also be an antibody, or an analogue of an antibody such as a single chain antibody, which binds the cell surface structure.
  • Ligands useful in forming the carrier will vary according to the particular cell to be targeted.
  • glycoproteins having exposed terminal carbohydrate groups such as asialoglycoprotein (galactose-terminal) can be used, although other ligands such as polypeptide hormones may also be employed.
  • asialoglycoproteins include asialoorosomucoid, asialofetuin and desialylated vesicular stomatitis virus.
  • Such ligands can be formed by chemical or enzymatic desialylation of glycoproteins that possess terminal sialic acid and penultimate galactose residues.
  • asialoglycoprotein ligands can be formed by coupling galactose terminal carbohydrates such as lactose or arabinogalactan to non-galactose bearing proteins by reductive lactosamination.
  • the cell-specific binding agent can be a receptor or receptor-like molecule, such as an antibody which binds a ligand (e.g., antigen) the cell surface.
  • ligands e.g., antigen
  • the gene-binding agent complexes the gene to be delivered. Complexation with the gene must be sufficiently stable in vivo to prevent significant uncoupling of the gene extracellularly prior to internalization by the target cell. However, the complex is cleavable under appropriate conditions within the cell so that the gene is released in functional form. For example, the complex can be labile in the acidic and enzyme rich environment of lysosomes. A noncovalent bond based on electrostatic attraction between the gene-binding agent and the expressible gene provides extracellular stability and is releasable under intracellular conditions.
  • Preferred gene-binding agents are polycations that bind negatively charged polynucleotides. These positively charged materials can bind noncovalently with the gene to form a soluble, targetable molecular complex which is stable extracellularly but releasable intracellularly.
  • Suitable polycations are polylysine, polyarginine, polyornithine, basic proteins such as histones, avidin, protamines and the like.
  • a preferred polycation is polylysine (e.g., ranging from 3,800 to 60,000 daltons).
  • noncovalent bonds that can b used to releasably link the expressible gene include hydrogen bonding, hydrophobic bonding electrostatic bonding alone or in combination such as, anti-polynucleotide antibodies bound to polynucleotide, and strepavidin or avidin binding to polynucleotide containing biotinylate nucleotides.
  • the carrier can be formed by chemically linking the cell-specific binding agent and the gene-binding agent.
  • the linkage is typically covalent.
  • a preferred linkage is a peptide bond. This can be formed with a water soluble carbodiimide as described by Jung, G. ej a (1981) Biochem. Bioshys. Res. Commun. 1Q .-.599-606.
  • An alternative linkage is a disulfid bond.
  • the linkage reaction can be optimized for the particular cell-specific binding agent and gene-binding agent used to form the carrier. Reaction conditions can be designed to maximize linkage formation but to minimize the formation of aggregates of the carrier components.
  • the optimal ratio of cell-specific binding agent to gene-binding agent can be determined empirically. When polycations are used, the molar ratio of the components will vary with the size of the polycation and the size of the gene- binding agent. In general, this ratio ranges from about 10:1 to 1:1, preferably about 5:1. Uncoupled components and aggregates can be separated from the carrier by molecular sieve or ion exchange chromatography (e.g., AquaporeTM cation exchange, Rai in).
  • asialoorosomucoid-polylysine conjugate is formed with the crosslinking agent l-(3-dimethylaminopropyl)-3-ethyl carbodiimide. After dialysis, the conjugate is separated from unconjugated components by preparative acid-urea polyacrylamide gel electrophoresis (pH 4-5). The conjugate can be further purified on the carboxymethyl functionalized column (Waters AP-1 column). See U.S. Patent Application Serial No. 08/043,008, filed on April 5, 1992, the teachings of which are incorporated by reference herein.
  • the gene encoding the antisense construct can be complexed to the carrier by a stepwise dialysis procedure.
  • the dialysis procedure begins with a 2M NaCl dialyzate and ends with .15M NaCl solution.
  • the gradually decreasing NaCl concentration results in binding of th gene to the carrier.
  • dialysis may not be necessary; the gene and carrier are simply mixed and incubated.
  • the molecular complex can contain more than one copy of the same gene or one or more different genes.
  • the weight ratio of gene to the carrier is from about 1 :5 t 5:1, preferably about 1:2 (approximate molar ratio 1:100 to 1:200).
  • the appropriate ratio f a particular polynucleotide and carrier can be determined by the gel retardation assay described in U.S. Patent No. 5,166,320.
  • the molecular complex of this invention can be administered parenterally. Preferably, it is injected intravenously.
  • the complex is administered in solution in a physiologically acceptable vehicle.
  • Cells can be transfected in vivo for transient production of the polyribonucleotide.
  • the gene can be administered repeatedly.
  • the transfected target cell can be stimulated to replicate by surgical or pharmacological means t prolong the activity of the incorporated gene.
  • Drugs that disrupt translocation or fusion of endosomes to lysosomes suc as colchicine or taxol can be used to prolong expression. See U.S. Patent Application Seria No. 950,789, filed September 24, 1992, the teachings of which are incorporated by referenc herein.
  • Delivery of the gene can be enhanced by coupling the carrier to a virus such as adenovirus.
  • a virus such as adenovirus.
  • the method of this invention can be used to selectively deliver a gene to a target cel in vivo for antisense gene therapy or other applications which require inhibition of the expression of specific cellular or foreign genes.
  • the RNA transcript produced from the delivered gene hybridizes with its complementary RNA, inhibiting its function either by steric hindrance, or by physical cleavage, thereby blocking expression of the target gene or genes.
  • the gene can be targeted to specific cells to alleviate a genetic abnormality caused by overexpression of a cellular or viral oncogene.
  • the method can be used to treat negative dominant genetic diseases in which an abnormal gene product interferes with a normal protein.
  • the method can be used to deliver a antisense or ribozyme directed against the abnormal fibrillin produced in Marfan's syndrom or against the abnormal collagen produced in osteogenesis imperfecta type I.
  • the method c also be used to inhibit the expression of the genes of an infecting pathogen such as a virus (hepatitis, HIV) or a parasite such as malaria, trypanosome, lysteria, or mycoplasma.
  • hepatitis genes such as the genes encoding one or more of the surface or core antigens can be assembled in an expression vector in reverse orientation to generate an antisense transcript which blocks translation of the corresponding genes.
  • the molecular complex of this invention is adaptable for delivery of a wide range of genes to a specific cell or tissue.
  • the complex is targeted to the liver by exploiting the hepatic asialoglycoprotein receptor system which allows for m vivo transfection of hepatocytes by the process of receptor-mediated endocytosis.
  • the method of the invention can be used to treat virus infections of liver cells. This includes infections by any of the liver-specific hepatitis viruses. Infection by human hepatiti B virus often results in a chronic, persistent infection. This form of viral infection is more suitably treated by antisense gene therapy compared to antisense oligonucleotide therapy. Because the hepatitis B virus genome is very small, coding for only three extensively overlapping RNA transcripts, antisense genes can be engineered to encode an RNA that can hybridize to one or more large regions common to all of the viral transcripts.
  • a complex can be used to deliver a plasmid-borne antisense gene specifically to chronically infected hepatocytes to block the production of hepatitis B virus.
  • the gene can code for the production of an antisense RNA transcript that hybridizes to all of the RNAs produced by the hepatitis B virus, inhibiting production of all viral polypeptides.
  • the resulting soluble complex is administered parenterally to target liver cells of the individual afflicted with the virus in amounts sufficient to selectively transfect the cells and to provide sufficient production of the antisense RNA to achieve inhibition of virus production.
  • the 9.4 Kb plasmid pAdw-HTD which contains two head-to-tail copies of the hepatitis B virus (HBV) genome, was provided by T.J. Liang (Massachusetts General
  • the first construct termed pJ3 ⁇ 1.0HTDl or "Anti S” was created by first digesting pAdw-HTD with the restriction endonucleases EcoRI and EcoRV followed by subsequent isolation of 1044bp fragment by agarose gel electrophoresis and glass bead extraction (Geneclean II®, BiolOl, LaJolla, CA). This fragment spans most of the pre-S2 signal peptide gene and the complete surface antigen gene but not the surface antigen promoter region. This EcoRI/EcoRV fragment was then ligated into the EcoRI and Smal sites within the polylinker region of the expression vector PJ3 ⁇ (ATCC, 37719, Nuc. Acids Res.
  • the second construct termed pJ3 ⁇ 0.8HTD3 or "Anti C” was designed to generate a 0.8 Kb antisense mRNA complementary to the region in the HBV pregenomic mRNA whic encodes the precore and core antigens.
  • Core antigen like the surface antigen, is a coat protein that is essential for viral packaging.
  • DR1 the 1 lb direct repeat sequence
  • DR1 the 1 lb direct repeat sequence
  • pJ3 ⁇ 0.8HTD3 pAdw-HTD was cleaved with the restriction endonucleases Fspl and Apal to generate a 802bp fragment which was separated by agarose gel electrophoresis and isolated using Geneclean II®.
  • the Anti C fragment was ligated into the Apal/Smal sites within the polylinker region of the cloning vector pGEM-7zf(+) (Promega, Madison, WI). The Anti C fragment was then subcloned in the Clal and Smal sites within the polylinker region of the pJ3 ⁇ vector.
  • the pGEM clone was first cut with Apal and blunted by the addition of the large Klenow fragment of DNA polymerase I plus 200mM dNTP mixture. Blunting of the Apal end was followed by digestion with Clal to yield a 818bp fragment which was then ligated into the pJ3 ⁇ expression vector ( Figure 1).
  • Competent DH5 ⁇ E coli (Gibco BRL) were transformed with the plasmid construct according to standard protocols (Sambrook ej aL (1989) Molecular Cloning - A Laboratory Manual. Cold Spring Harbor Laboratory, 2nd ed.). Large scale preparations of the plasmid DNA were carried out by standard procedures.
  • HepG2 .2.15 Human hepatoma, HepG2 .2.15 cells (provided by Dr. George Acs, Albert Einstein College of Medicine, Bronx, NY) were maintained and grown in minimum essential maxim (MEM) supplemented with 0.1 mM non-essential amino acids, 0.1 mM sodiumpyruvate, 2m L-glutamine, 50 units/ml penicillin, 50 ⁇ g/ml streptomycin and 10% fetal calf serum.
  • HepG .2.15 are clonal cells derived from HepG2 cells transformed with a plasmid containing the HBV genome.
  • HepG2 .2.15 cells were seeded at 3.5xl0 ⁇ /60mm dish 24 hours prior to use. Preceding transfection, all cells were treated with 1 OO ⁇ M chloroquine for one hour, followed by 3 washes with phosphate-buffered saline.
  • hepatitis B virus surface antigen (HBsAg) levels To determine baseline hepatitis B virus surface antigen (HBsAg) levels, an ELISA (Abbott) assay was performed on 50 ⁇ l samples of medium removed from each plate prior to transfection and assayed according to the procedure described by the manufacturer. 50 ⁇ l samples of medium were subsequently removed from each dish every day for 6 days and processed for HBsAg.
  • RESULTS pJ3 ⁇ 0.8HTD3 was introduced into HepG 2.2.15 cells via asialoglycoprotein receptor mediated endocytosis in order to examine the plasmid's ability to inhibit production of HBsAg.
  • some cells were also treated with an antisense 21-mer oligodeoxynucleotide directed against the unique HBV polyadenylation signal sequence. This oligodeoxynucleotide was idential to the one used by Wu and Wu (1992) J. Biol. Chem. 267:12436-12439 to inhibit HepG 2.2.15 HBsAg expression.
  • each molecule of pJ3 ⁇ 0.8HTD3 was roughly equivalent to 1400 molecules of the antisense oligonucleotide i inhibiting HBsAg expression. We expect that this was due to the ability of the plasmid to generate many copies of its antisense transcript once it was delivered into the cell. It is als possible that differences in stability, site of action, intracellular retention, or differences in t properties of the complexes made with each type of DNA may have contributed to this effe This illustrates a potential advantage of plasmid based antisense systems compared to oligonucleotides. A smaller amount of DNA needs to be delivered to the target eel in order achieve a therapeutic dose.
  • a plasmid contains cis-acting sequences, such as promoters, enhances, polyadenylation sites, origins of replication, etc. which directed or influence the expression of its RNA.
  • cis-acting sequences such as promoters, enhances, polyadenylation sites, origins of replication, etc. which directed or influence the expression of its RNA.
  • These sequences can be modified or substituted in or to tailer expression for specific circumstances.
  • an inducible promoter into the plasmid in order to activate expression of the antisense mRNA only at certain times or under certain conditions.
  • consensus RNA destabilizing elements within the 3' untranslated region of the mRNA.
  • Another possibility would be to produce a sustained antisense mediated inhibition by incorporated sequences into the plasmid which would allo it to be maintained episomally within the cell.
  • Many other characteristics could also be incorporated into a plasmid-based antisense system, thus allowing for a great deal of flexibiltiy

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biotechnology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Sont décrits des complexes moléculaires permettant l'apport ciblé d'un gène codant un polyribonucléotide antisens à une cellule spécifique in vivo, l'obtention de la production du polyribonucléotide dans la cellule ciblée et l'inhibition spécifique de l'expression de gènes cellulaires ou non cellulaires. On forme un complexe avec un gène exprimable codant un polyribonucléotide antisens et un conjugué constitué d'un agent de liaison spécifique de la cellule et d'un agent de liaison du gène. L'agent de liaison spécifique de la cellule est spécifique d'une structure superficielle cellulaire qui sert de médiateur à l'internalisation de ligands par endocytose. Un exemple d'un tel agent est le récepteur asialoglycoprotéique d'hépatocytes. L'agent de liaison du gène est un composé tel qu'un polycation qui forme, de façon stable, le complexe comprenant le gène dans des conditions extracellulaires et libère le gène dans des conditions intracellulaires de sorte qu'il puisse fonctionner dans une cellule. Le complexe moléculaire selon l'invention est stable et soluble dans des liquides physiologiques, et il peut être utilisé dans la thérapie génique antisens pour transfecter, de façon sélective, des cellules in vivo ou in vitro, cela afin de produire le polyribonucléotide antisens dans la cellule ciblée, et d'inhiber l'expression de gènes cellulaires ou non cellulaires.
EP94912389A 1993-04-05 1994-04-04 Apport cible de genes codant des polyribonucleotides antisens Withdrawn EP0693130A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US4294393A 1993-04-05 1993-04-05
US42943 1993-04-05
PCT/US1994/003643 WO1994023050A1 (fr) 1993-04-05 1994-04-04 Apport cible de genes codant des polyribonucleotides antisens

Publications (1)

Publication Number Publication Date
EP0693130A1 true EP0693130A1 (fr) 1996-01-24

Family

ID=21924584

Family Applications (1)

Application Number Title Priority Date Filing Date
EP94912389A Withdrawn EP0693130A1 (fr) 1993-04-05 1994-04-04 Apport cible de genes codant des polyribonucleotides antisens

Country Status (5)

Country Link
EP (1) EP0693130A1 (fr)
JP (1) JPH08508645A (fr)
AU (1) AU686614B2 (fr)
CA (1) CA2159916A1 (fr)
WO (1) WO1994023050A1 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU4690596A (en) * 1994-12-30 1996-07-24 Chiron Viagene, Inc. Nucleic acid condensing agents with reduced immunogenicity
US5780009A (en) * 1995-01-20 1998-07-14 Nexia Biotechnologies, Inc. Direct gene transfer into the ruminant mammary gland
US6673914B1 (en) 1998-01-22 2004-01-06 John Wayne Cancer Institute Human tumor-associated gene
AU1480801A (en) * 1999-11-12 2001-06-06 Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The Compositions and methods for the diminution or elimination of various cancers
US7927792B2 (en) 2002-11-18 2011-04-19 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Targeted double stranded RNA mediated cell killing
WO2008039980A2 (fr) 2006-09-28 2008-04-03 Loma Linda University Transfection apoptotique à médiation cellulaire de cellules de mammifère par arn interférant
JP6245570B2 (ja) * 2013-10-08 2017-12-13 国立研究開発法人理化学研究所 トリパノソーマ関連疾患治療薬、トリパノソーマ原虫の殺虫方法およびその利用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992005250A1 (fr) * 1990-09-25 1992-04-02 University Of Connecticut Prolongation de l'expression de polynucleotides introduits dans une cellule
WO1992006693A1 (fr) * 1990-10-22 1992-04-30 Fox Chase Cancer Center Produit de recombinaison d'adn destine a la therapie par l'arn
KR100252547B1 (ko) * 1991-09-05 2000-09-01 프레드 마리얀스키 폴리-또는 올리고누클레오티드의 세포로의 표적화된 전달

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9423050A1 *

Also Published As

Publication number Publication date
WO1994023050A1 (fr) 1994-10-13
AU686614B2 (en) 1998-02-12
AU6497894A (en) 1994-10-24
JPH08508645A (ja) 1996-09-17
CA2159916A1 (fr) 1994-10-13

Similar Documents

Publication Publication Date Title
AU704562C (en) Anti-hepatitis B poly- and oligonucleotides
CN103547272B (zh) 基于肽的体内siRNA递送系统
Moschos et al. Lung delivery studies using siRNA conjugated to TAT (48− 60) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity
JP3316216B2 (ja) 安定化外部ガイド配列
AU683400B2 (en) Use of a bacterial component to enhance targeted delivery of polynucleotides to cells
US20170056507A1 (en) LOW DENSITY LIPOPROTEIN RECEPTOR-MEDIATED siRNA DELIVERY
US20100233141A1 (en) Compositions And Methods For The Delivery Of Biologically Active RNAs
US20040167090A1 (en) Covalent modification of RNA for in vitro and in vivo delivery
WO2015002956A1 (fr) Système de distribution d'exosome
US5843770A (en) Antisense constructs directed against viral post-transcriptional regulatory sequences
WO1994006923A1 (fr) Modification d'un virus pour rediriger l'infectivite et amplifier l'apport cible de polynucleotides a des cellules
US20170043012A1 (en) Chimeric therapeutics, compositions, and methods for using same
JP2002517180A (ja) 増大された発現を示す新規ベクターおよび遺伝子
EP2794879A1 (fr) Compositions et procédés pour l'administration d'arn biologiquement actifs
AU686614B2 (en) Targeted delivery of genes encoding antisense polyribonucleotides
JPH06503714A (ja) 細胞内に導入されたポリヌクレオチドの長時間発現
Madon et al. Receptor–Mediated Delivery of Hepatitis B Virus Dna and Antisense Oligodeoxynucleotides to Avian Liver Cells
US5744326A (en) Use of viral CIS-acting post-transcriptional regulatory sequences to increase expression of intronless genes containing near-consensus splice sites
KR20220117091A (ko) 생체내 세포에서 합성 및 분비되고 세포로 투과하는 관심 폴리펩타이드를 코딩하는 발현카세트 및 이의 용도
AU735667B2 (en) Targeted delivery of genes encoding secretory proteins
CN114767704A (zh) 一种能够靶向乙型肝炎病毒的药物构造及药物组合物
Shim et al. 7 Targeted Delivery Systems of Nonviral Nucleic Acid–Based Therapeutics
US20040009900A1 (en) Targeted delivery of genes encoding secretory proteins
WO1994022485A1 (fr) Apport cible de genes codant des enzymes microsomales cellulaires
WO1999015687A1 (fr) Complexe polynucleotidique multiconstituant pour transfection cellulaire a haute efficacite

Legal Events

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

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19951013

AK Designated contracting states

Kind code of ref document: A1

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

17Q First examination report despatched

Effective date: 19991130

GRAG Despatch of communication of intention to grant

Free format text: ORIGINAL CODE: EPIDOS AGRA

GRAG Despatch of communication of intention to grant

Free format text: ORIGINAL CODE: EPIDOS AGRA

GRAH Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOS IGRA

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

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20021204