WO1996002257A1 - Oligonucleotides antisens de pleiotrophine - Google Patents

Oligonucleotides antisens de pleiotrophine Download PDF

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Publication number
WO1996002257A1
WO1996002257A1 PCT/US1995/008781 US9508781W WO9602257A1 WO 1996002257 A1 WO1996002257 A1 WO 1996002257A1 US 9508781 W US9508781 W US 9508781W WO 9602257 A1 WO9602257 A1 WO 9602257A1
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oligonucleotide
pleiotrophin
cells
ptn
cell
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PCT/US1995/008781
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English (en)
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Kenneth J. Colley
Anton Wellstein
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Georgetown University
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Priority to AU31265/95A priority Critical patent/AU3126595A/en
Priority to JP8505138A priority patent/JPH10506003A/ja
Publication of WO1996002257A1 publication Critical patent/WO1996002257A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N15/1136—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 growth factors, growth regulators, cytokines, lymphokines or hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • 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
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • Polypeptide growth factors have been shown to play important physiological roles in the timely development of tissues during embryonal and neonatal growth and, therefore, their expression is tightly regulated. Conversely, polypeptide growth factor gene expression is deregulated in tumor cell lines, as well as in solid tumors, and the activity of the corresponding growth factors appears to contribute significantly to autocrine and paracrine stimuli. Cross and Dexter, Cell 64:271 (1991) .
  • Pleiotrophin is an 18 D heparin binding protein originally purified as a weak mitogen from bovine uterus and as a neurite outgrowth promoter from neonatal rat brain. Milner et al . , Biochem. Bi ⁇ phys . Res . Commun . 165:1096-1103 (1989); Rauvala, EMBO J. 8:2933-2941 (1989); Li et al. , Science 250:1690-1694 (1990). PTN appears to belong to a family of heparin binding growth factors. Lai et al . , Biochem. Biophys . Res . Commun . 187:1113-1121 (1992).
  • the cDNA's for human, bovine and rat PTN's have been cloned and sequenced and shown to exhibit sequence identity with a retinoic acid-induced differentiation factor and retinoic acid-induced heparin binding protein from chicken embryo.
  • PTN transcripts are expressed in a restricted pattern within tissue and are highly regulated during murine development.
  • PTN and the closely related midkine (MK) proteins appear to play a role during development of the neuroectoderm, and the physiologic expression of the genes in the adult occurs only in very restricted areas of the nervous system.
  • MK midkine
  • PTN also has been linked to cancer formation. For example, expression of PTN is elevated in melanomas that are highly vascularized, and PTN supports the growth of SW13 cells in soft agar. Wellstein et al . , J. Biol . Chem. 267:2582-2587 (1992).
  • PTN expression can induce tumors to grow in nude mice, and high levels of PTN mRNA are detected in tissue samples from human breast cancers. Fang et al . , J. Biol . Chem. 267 25889-25897 (1992). In the same study, about one-fourth of tumor cell lines tested showed expression of PTN, as measured by RNase protection assays. Carcinogen-induced tumors in rat mammary tissue also scored positive for PTN expression. Fang et al. (1992) .
  • compositions that inhibit the expression of pleiotrophin It is a further object of this invention to provide compositions that inhibit the expression of pleiotrophin. It is yet a further object of this invention to provide antisense oligonucleotides which inhibit pleiotrophin expression by controlling translation of the mRNA corresponding to the pleiotrophin gene.
  • a method for inhibiting expression of PTN in a cell by introducing an oligonucleotide that is capable of hybridizing to the single-stranded mRNA encoding human pleiotrophin.
  • the PTN is a human PTN.
  • the oligonucleotide is contained in a liposome.
  • composition comprising at least one antisense oligonucleotide that, when introduced into a host cell, binds to a segment of a single-stranded mRNA transcribed from a pleiotrophin gene, and that inhibits pleiotrophin synthesis in said cell.
  • a pharmaceutically useful preparation comprising at least one PTN antisense oligonucleotide in a pharmaceutically acceptable sterile vehicle.
  • a method for treating a pathological growth of tissue comprising the step of inhibiting expression of a pleiotrophin gene.
  • the pathological growth is a dysplastic or neoplastic disorder.
  • a method for treating a pathological growth of tissue in a patient comprising administering to said patient an amount of at least one PTN antisense oligonucleotide sufficient to inhibit pleiotropin synthesis in said patient.
  • FIGURE 1 depicts the cDNA sequence (SEQ ID NO:l) for human pleiotrophin.
  • FIGURE 2 depicts the location of the antisense primers within the cDNA sequence for human pleiotrophin (SEQ ID NO:2) .
  • the present invention involves methods for the inhibition of the synthesis of pleiotrophin, thus providing a therapeutic regimen for the treatment of neoplasias and dysplasias.
  • the invention is based on the use of antisense oligonucleotides which anneal to pleiotrophin-specific single-stranded RNA, and which thereby inhibit production of pleiotrophin. Inhibition of pleiotrophin synthesis represses the corresponding growth-stimulating activity and alleviates neoplastic and dysplastic conditions associated with PTN.
  • oligonucleotides are provided that are designed to be hybridize to portions of the mRNA coding for pleiotrophin, thereby disrupting the functions of these
  • the present invention also includes pharmaceutical compositions comprising an effective amount of at least one of the antisense oligonucleotides of the invention in combination with a pharmaceutically acceptable sterile vehicle, as described in Remingtons's Pharmaceutical Sciences; Drug Receptors and Receptor Theory, 18th ed. , Mack Publishing Co., Easton, PA (1990) .
  • Antisense technology offers a very specific and potent means of inhibition of this gene product. See Stein and Chang, Science 261:1004-12 (1993) .
  • Antisense oligonucleotides (“antisense oligos") are typically short sequences of DNA, usually 10-50 bases in length, that are complementary to specific regions of a corresponding target mRNA.
  • Hybridization of antisense oligos to their target transcripts is highly specific as a result of complementary base pairing. Hybridization of antisense oligos is affected by such parameters as length, chemical modification and secondary structure of the transcript which can influence oligo access to the target site. See Stein et al , Cancer Research 48:2659 (1988).
  • oligos such as 10-to 15-mers, while offering higher cell penetration, have lower gene specificity. In contrast, while longer oligos of 20-30 bases offer better specificity, they show decreased uptake kinetics into cells. See Stein et al . , PHOSPHOROTHIOATE OLIGODEOXYNUCLEOTIDE ANALOGUES in "Oligodeoxynucleotides - Antisense Inhibitors of Gene Expression" Cohen, ed. McMillan Press, London (1988) . Accessibility to mRNA target sequences also is of importance and, therefore, loop-forming regions in targeted mRNAs offer promising targets.
  • oligonucleotide encompasses both oligomeric nucleic acid moieties of the type found in nature, such as the deoxyribonucleotide and ribonucleotide structures of DNA and RNA, and man-made analogues which are capable of binding to nucleic acids found in nature.
  • the oligonucleotides of the present invention can be based upon ribonucleotide or deoxyribonucleotide monomers linked by phosphodiester bonds, or by analogues linked by methyl phosphonate, phosphorothioate, or other bonds.
  • oligonucleotides may also comprise monomer moieties which have altered base structures or other modifications, but which still retain the ability to bind to naturally occurring DNA and RNA structures.
  • Such oligonucleotides may be prepared by methods well- known in the art, for instance using commercially available machines and reagents available from Perkin- Elmer/Applied Biosystems (Foster City, CA) .
  • Phosphodiester-linked oligonucleotides are particularly susceptible to the action of nucleases in serum or inside cells, and therefore in a preferred embodiment the oligonucleotides of the present invention are phosphorothioate or methyl phosphonate-linked analogues, which have been shown to be nuclease- resistant. See Stein et al .
  • the antisense oligonucleotide is an RNA molecule produced by transfection of the target cell with an expression construct.
  • the RNA molecule thus produced is chosen to hybridize to pleiotrophin mRNA, thus inhibiting translation of the mRNA and inhibiting pleiotrophin synthesis.
  • Hybridization of the oligos with mRNA targets can inhibit expression of corresponding gene products by multiple mechanisms. In “translation arrest,” the interaction of oligos with target mRNA blocks the action of the ribosomal complex and, hence, prevents translation of the messenger RNA into protein.
  • “translation arrest” the interaction of oligos with target mRNA blocks the action of the ribosomal complex and, hence, prevents translation of the messenger RNA into protein.
  • oligonucleotides can form "triplex, " or triple-helical structures with double stranded genomic DNA containing the gene of interest, thus interfering with transcription by RNA polymerase. Giovannangeli et al . , Proc . Natl . Acad. Sci . 90:10013 (1993); Ebbinghaus et al . J. Clin . Invest . 92:2433 (1993).
  • PTN oligonucleotides are synthesized according to standard methodology. Phosphorothioate modified DNA oligonucleotides typically are synthesized on automated DNA synthesizers available from a variety of manufacturers.
  • oligos may be purified by polyacrylamide gel electrophoresis or reverse phase chromatography. See Sambrook et al . , MOLECULAR CLONING:
  • PTN oligonucleotides in the form of antisense RNA may be expressed transiently in appropriate cells from standard DNA expression vectors.
  • PTN DNA sequences can be cloned from standard plasmids into expression vectors, which expression vectors have characteristics permitting higher levels of, or more efficient expression of the resident oligonucleotides.
  • these constructs require a prokaryotic or eukaryotic promoter sequence which initiates transcription of the inserted DNA sequences.
  • a preferred expression vector is one where the expression is inducible to high levels. This is accomplished by the addition of a regulatory region which provides increased transcription of downstream sequences in the appropriate host cell. See Sambrook et al . , Vol. 3, Chapter 16 (1989) .
  • PTN antisense expression vectors can be constructed using the polymerase chain reaction (PCR) to amplify appropriate fragments from single- stranded cDNA of plasmid pRc-PTN.
  • PCR polymerase chain reaction
  • Figure 2 discloses nucleotide sequences of suitable oligonucleotide primers for the PCR reaction. Oligonucleotide synthesis and purification techniques are described in Sambrook et al . and Ausubel et al .
  • PCR procedures are performed via well-known methodology. See, for example, Ausubel, and Bangham, "The Polymerase Chain Reaction: Getting Started, " in PROTOCOLS IN HUMAN MOLECULAR GENETICS (Humana Press 1991) . Moreover, PCR kits can be purchased from companies such as Stratagene Cloning Systems (La Jolla, CA) and Invitrogen (San Diego, CA) .
  • cloning vector is a DNA molecule, such as a plasmid, cosmid or bacteriophage, that can replicate autonomously in a host prokaryotic cell.
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of an essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector.
  • Marker genes typically include genes that provide tetracycline resistance or a picillin resistance.
  • Suitable cloning vectors are described by Sambrook et al . , Ausubel, and Brown (ed.), MOLECULAR BIOLOGY LABFAX
  • Cloning vectors can be obtained, for example, from GIBCO/BRL (Gaithersburg, MD) , Clontech
  • the PCR products are ligated into a "TA" cloning vector.
  • TA cloning vector.
  • Methods for generating PCR products with a thymidine or adenine overhang are well- known to those of skill in the art. See, for example, Ausubel at pages 15.7.1-15.7.6.
  • kits for performing TA cloning can be purchased from companies such as Invitrogen (San Diego, CA) .
  • Cloned antisense fragments are amplified by transforming competent bacterial cells with a cloning vector and growing the bacterial host cells in the presence of the appropriate antibiotic. See, for example, Sambrook et al . , and Ausubel. PCR is then used to screen bacterial host cells for PTN antisense orientation clones. The use of PCR for bacterial host cells is described, for example, by Hofmann et al., "Sequencing DNA Amplified Directly from a Bacterial Colony, " in PCR PROTOCOLS: METHODS AND APPLICATIONS, White (ed.), pages 205-210 (Humana Press 1993), and by Cooper et al . , "PCR- Based Full-Length cDNA Cloning Utilizing the Universal- Adaptor/Specific DOS Primer-Pair Strategy, " Id. at pages 305-316.
  • Cloned antisense fragments are cleaved from the cloning vector and inserted into an expression vector.
  • an expression vector For example, HindiII and Xbal can be used to cleave the antisense fragment from TA cloning vector pCRTM-II (Invitrogen;San Diego, CA) .
  • Suitable expression vectors typically contain (1) prokaryotic DNA elements coding for a bacterial origin of replication and an antibiotic resistance marker to provide for the amplification and selection of the expression vector in a bacterial host; (2) DNA elements that control initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of transcripts, such as a transcription termination/polyadenylation sequence.
  • the transcriptional and translational regulatory signals preferably are derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression.
  • viral sources such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression.
  • Suitable transcriptional and translational regulatory sequences also can be obtained from mammalian genes, such as actin, collagen, myosin, and metallothionein genes.
  • Transcriptional regulatory sequences include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Suitable eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer et al . , J. Molec. Appl . Genet . 1: 273 (1982)) ; the TK promoter of Herpes virus (McKnight, Cell 31: 355 (1982)); the SV40 early promoter (Benoist et al . , Nature 290: 304
  • Rous sarcoma virus promoter Gorman et al . , Proc . Nat ' l Acad. Sci . USA 79: 6777 (1982)); and the cytomegalovirus promoter (Foecking et al . , Gene 45: 101 (1980) ) .
  • a prokaryotic promoter such as the bacteriophage T3 RNA polymerase promoter, can be used to control fusion gene expression if the prokaryotic promoter is regulated by a eukaryotic promoter.
  • a prokaryotic promoter such as the bacteriophage T3 RNA polymerase promoter
  • a suitable vector for expression in mammalian cells is the vector pRc/CMV (Invitrogen (San Diego, CA) , which provides a high level of constitutive transcription from mammalian enhancer-promoter sequences.
  • Cloned PTN antisense vectors are amplified in bacterial host cells, isolated from the cells, and analyzed as described above. Another possible method by which antisense sequences may be exploited is via gene therapy.
  • Virus- like vectors usually derived from retroviruses, may prove useful as vehicles for the importation and expression of antisense constructs in tumor cells. Generally, such vectors are non-replicative in vivo, precluding any unintended infection of non-target cells.
  • helper cell lines are provided which supply the missing replicative functions in vi tro, thereby permitting amplification and packaging of the antisense vector.
  • a further precaution against accidental infection of non-tumor cells involves the use of tumor cell-specific regulatory sequences. When under the control of such sequences, antisense constructs would not be expressed in normal tissues.
  • the antisense oligonucleotide effectively inhibited androgen-induced DNA synthesis.
  • Morrison showed that antisense oligonucleotides targeted against basic fibroblast growth factor can inhibit growth of astrocytes in culture.
  • the general feasibility of targeting tumor-related growth factors has been established.
  • Antisense oligonucleotides according to the present invention are derived from any portion of the open reading frame of the pleiotrophin cDNA. Preferably, mRNA sequences (i) surrounding the translation initiation site and (ii) forming loop structures are targeted. Based upon the size of the human genome, statistical studies show that a DNA segment approximately 14-15 base pairs long will have a unique sequence in the genome. To ensure specificity of targeting pleiotrophin RNA, therefore, it is preferred that the antisense oligonucleotides are at least 14 nucleotides in length, and preferably 15 nucleotides in length.
  • oligonucleotides contemplated by the present invention encompass nucleotides corresponding to positions 1-14, 1- 15, 1-16, 1-17, 1-18, 1-19, 2-16, 3-17, etc. of the pleiotrophin cDNA sequence. All possible oligonucleotides are represented by nucleotides according to the formula n to n + x, where n is 1 to 1383 and x is 14, 15, 16, 17, 18 or 19.
  • PTN activity it is possible to assay for PTN activity directly.
  • cell lines are available which have elevated levels of PTN. These cells also are characterized by certain behavioral abnormalities such as soft agar colony formation.
  • the increased proliferation of endothelial cells can be measured, and this is a useful in vi tro model for angiogenesis in vivo.
  • Treatment of such cells with effective antisense oligos will result in the alteration of the cell's behavior and serve to identify useful oligos.
  • Assays such as those described above serve as standard models for tumor growth in the body. Antisense oligonucleotides can be tested for in vivo efficacy and safety in an animal model system.
  • a preferred animal model is one in which the animal bears tumors as closely related as possible to those found in humans.
  • the mouse is a nude, athymic mouse carrying explanted human tumor cells which will produce clinical symptoms analogous to those observed in human cancer.
  • Such a mouse is a standard animal model used in the development of chemotherapeutic drugs. See, for example, Pitot, "Fundamentals of Oncology” 3rd ed., Marcel Dekker, Inc., New York, 1986, at 452.
  • an antisense oligonucleotide to a subject can be effected via any common route (oral, nasal, buccal, rectal, vaginal, or topical) , or by subcutaneous, intramuscular, intraperitoneal, or intravenous injection.
  • Pharmaceutical compositions of the present invention are advantageously administered in the form of injectable compositions.
  • a typical composition for such purpose comprises a pharmaceutically acceptable solvent or diluent and other suitable, physiologic compounds.
  • the composition may contain oligonucleotide and about 10 mg of human serum albumin per milliliter of a phosphate buffer containing NaCl.
  • antisense oligodeoxynucleotide As much as 700 milligrams of antisense oligodeoxynucleotide has been administered intravenously to a patient over a course of 10 days (i.e., 0.05 mg/kg/hour) without signs of toxicity.
  • Other pharmaceutically acceptable excipients include non-aqueous or aqueous solutions and non-toxic compositions including salts, preservatives, buffers and the like. Examples of non-aqueous solutions are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate.
  • Aqueous solutions include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.
  • Intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases.
  • the pH and exact concentration of the various components the pharmaceutical composition are adjusted according to routine skills in the art.
  • a preferred pharmaceutical composition for topical administration is a dermal cream or transdermal patch.
  • Antisense oligonucleotides or antisense expression vectors may be administered by injection as an oily suspension.
  • Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides.
  • antisense oligonucleotides or vectors may be combined with a lipophilic carrier such as any one of a number of sterols including cholesterol, cholate and deoxycholic acid.
  • a preferred sterol is cholesterol.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension also contains stabilizers.
  • Liposomes are microscopic vesicles that consist of one or more lipid bilayers surrounding aqueous compartments. See, generally, Bakker-Woudenberg et al . , Eur. J. Clin . Microbiol . Infect . Dis . 12 (Suppl. 1): S61 (1993), and Kim, Drugs 46: 618 (1993) . Liposomes are similar in composition to cellular membranes and as a result, liposomes can be administered safely and are biodegradable.
  • liposomes may be unilamellar or multilamellar, and liposomes can vary in size with diameters ranging from 0.02 ⁇ m to greater than 10 ⁇ m.
  • agents can be encapsulated in liposomes: hydrophobic agents partition in the bilayers and hydrophilic agents partition within the inner aqueous space(s). See, for example, Machy et al . , LIPOSOMES IN CELL BIOLOGY AND PHARMACOLOGY (John Libbey 1987), and Ostro et al . , American J. Hosp . Pharm. 46: 1576 (1989) .
  • it is possible to control the therapeutic availability of the encapsulated agent by varying liposome size, the number of bilayers, lipid composition, as well as the charge and surface characteristics of the liposomes.
  • Liposomes can adsorb to virtually any type of cell and then slowly release the encapsulated agent.
  • an absorbed liposome may be endocytosed by cells that are phagocytic. Endocytosis is followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents. Scherphof et al . , Arm. N. Y. Acad. Sci . 446: 368 (1985).
  • reticuloendothelial system After intravenous administration, conventional liposomes are preferentially phagocytosed into the reticuloendothelial system.
  • the reticuloendothelial system can be circumvented by several methods including saturation with large doses of liposome particles, or selective macrophage inactivation by pharmacological means. Claassen et al., Biochim. Biophys . Acta 802: 428 (1984) .
  • incorporation of glycolipid- or polyethelene glycol- derivatised phospholipids into liposome membranes has been shown to result in a significantly reduced uptake by the reticuloendothelial system. Allen et al . , Biochim. Biophys .
  • Antisense oligonucleotides and expression vectors can be encapsulated within liposomes using standard techniques.
  • a variety of different liposome compositions and methods for synthesis are known to those of skill in the art. See, for example, U.S. Patent No. 4,844,904, U.S. Patent No. 5,000,959, U.S. Patent No. 4,863,740, and U.S. Patent No. 4,975,282, all of which are hereby incorporated by reference.
  • Liposomes can be prepared for targeting to particular cells or organs by varying phospholipid composition or by inserting receptors or ligands into the liposomes.
  • antibodies specific to tumor associated antigens may be incorporated into liposomes, together with antisense oligonucleotides or expression vectors, to target the liposome more effectively to the tumor cells. See, for example, Zelphati et al . , Antisense Research and Development 3: 323-338 (1993) , describing the use "immunoliposomes" containing antisense oligonucleotides for human therapy.
  • the dosage of administered liposome- encapsulated antisense oligonucleotides and vectors will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. Dose ranges for particular formulations can be determined by using a suitable animal model.
  • Growths may be dysplastic, i . e . , abnormal tissue growth that is benign in character, such as retinopathies, arthritis, psoriasis, nevi and virally-induced dysplasias. Growths may also be neoplastic, i . e . , associated with tumor formation and malignancy, such as melanoma, breast cancer, ovarian cancer, prostate cancer, glioblastoma, neuroblastoma and metastatic disease EXAMPLE 1.
  • dysplastic i . e .
  • abnormal tissue growth that is benign in character, such as retinopathies, arthritis, psoriasis, nevi and virally-induced dysplasias.
  • Growths may also be neoplastic, i . e . , associated with tumor formation and malignancy, such as melanoma, breast cancer, ovarian cancer, prostate cancer, glioblastoma, neuroblastoma and metastatic disease EX
  • SW-13 cells human adrenal carcinoma
  • ATCC American Type Culture Collection
  • SW-13/PTN cells are SW-13 cells stably transfected with a PTN- containing clone to overexpress human pleiotrophin, as previously described.
  • SW-13/MK cells are SW-13 cells transfected to overexpress the human midkine gene. Sale et al . , manuscript in preparation (1994) .
  • SW-13 cell lines were maintained in improved minimum essential medium (IMEM; Biofluids Inc., Rockville, MD) with 10% fetal bovine serum (FBS; Biofluids Inc.).
  • Human metastatic melanoma cells (1205- LU) were a gift from Dr. M. Herlyn of the Wistar Institute, Philadelphia, PA and were maintained in media containing 80% keratinocyte serum free medium (KSFM; GIBCO/BRL, Bethesda, MD) and 20% Leibowitz medium (L-15; GIBCO/BRL) with 5% FBS and 1.5 mM CaCl 2 .
  • KSFM keratinocyte serum free medium
  • L-15 Leibowitz medium
  • huBEC Human brain endothelial cells
  • the first antisense oligo was complementary to the translation initiation codon and the second was complementary to a loop-forming region in the open reading frame of PTN.
  • ptnASl SEQ ID N0:1
  • ptnSCRl SEQ ID NO:2
  • ptnAS3 SEQ ID NO: 3
  • ptnSCR3 SEQ ID NO:4
  • the oligos were solubilized in PBS before addition to cells and their concentrations verified by optical density .
  • ELISA Assays Conditioned media was removed from the treated cells at the specified times and filtered through low protein binding membranes to remove debris before plating 100-200 ⁇ l at serial dilutions in 96-well plates (MaxiSorp; Nunc, Thomas Scientific, Swedesboro, NJ) . After plating, the wells were allowed to dry. Alternatively, the conditioned media was concentrated and partially purified by heparin-affinity chromatography before plating. Wellstein et al. , (1992) .
  • Each well was washed three times with 200 ⁇ l of phosphate buffered saline-0.5% Tween-20 wash solution (PBS/Tween) before each step and four times before the final step.
  • PBS/Tween phosphate buffered saline-0.5% Tween-20 wash solution
  • Each well was treated with 100-200 ⁇ l PBS/Tween with 1% BSA for one hour at room temperature to block non-specific binding before addition of 100 ⁇ l primary PTN antibody at 1:500 diultion (PTN-1 rabbit antisera raised by this laboratory; PTN-HBNF rabbit antisera, the gift of Dr. P. B ⁇ hlen, Lederle Laboratories, Pearl River, N.Y.
  • PTN-HBNF rabbit antisera the gift of Dr. P. B ⁇ hlen, Lederle Laboratories, Pearl River, N.Y.
  • the primary MK antibody MK rabbit antisera raised by this laboratory was diluted 1:1000.
  • Soft Agar Assays and Cocul ture Experiments . Formation of colonies in soft agar by SW-13, SW-13/PTN or SW-13/MK cells was determined as described previously. Wellstein et al . (1990) . In brief, 20,000 cells in 0.35% agar (Bactoagar; GIBCO/BRL) were layered on top of 1 ml of a solidified 0.6% agar layer in a 35 mm dish (Costar Corp. Cambridge) . Material to be tested was filter- sterilized, 500 ⁇ l of which was added with the 800 ⁇ l top layer unless indicated otherwise. Growth media with 10% FBS was included in both layers. Colonies more than 60 ⁇ m in diameter were counted after 1-2 weeks of incubation at 37°C using an image analyzer.
  • Conditioned media from PTN-expressing cells was added to SW-13 cells.
  • Antisense oligos as indicated in the text, were added to SW-13/PTN or SW-13/MK cells.
  • coculture of PTN-expressing cells and SW- 13 cells was studied in a similar manner, except that the PTN-expressing cells were first plated on the bottom of 35 mm dishes at densities of 1 X 10 3 to 5 X 10 3 cells per dish, followed by addition of the agar layers and SW-13 cells. During the interval before the addition of agar and SW-13 cells, the PTN-expressing cells were allowed to adhere to the plastic and were treated with antisense oligos for the specified time and concentration.
  • huBEC cells were plated at a density of 5 X 10 3 per well in the bottom of a six-well culture plate (Falcon, Becton Dickinson, Franklin Lakes, NJ) . 1205-LU cells were plated at a density of 5 X 10 4 in 0.45 ⁇ m porosity cell culture inserts (Falcon) and treated for 72 hours with either antisense oligo, control oligo (scrambled sequence) or phosphate buffered saline (PBS) before transferring the inserts into the huBEC culture plates for continued coculture of one week. The huBEC cells were then detached with trypsin and counted with a particle counter.
  • conditioned media from oligo- treated 1205-LU cells was harvested and added to the huBECs in culture at different concentrations. Cell numbers were counted after six days. Tumor Growth in Animals . Pre-confluent melanoma cells were pretreated with the specified concentration of either antisense oligo DNA, control oligo DNA or PBS for 72 hours. After this time period, cells were trypsinized from the treatment flasks and washed three times in melanoma media before collection.
  • the antisense oligos were selected for higher G+C content at their ends to improve hybridization characteristics. Stein et al , loc. ci t . (1989) .
  • the control oligos were chemically-identical, scrambled sequences (ptnSCRl and ptnSCR3, SEQ ID NOS 2 and 4) of the respective antisense oligos. It was determined that these oligos did not have a significant antisense relationship to other regions in PTN or in other genes.
  • Sense oligos were avoided as control sequences due to the fact that they are usually chemically different from the respective antisense oligos in as much as they contain different compositions of nucleotides.
  • SW-13 cells that had been transfected with a PTN expression vector. These cells form colonies in agar as a result of PTN activity. Fang et al . (1992) . In these experiments, SW-13/PTN cell colony formation was inhibited by 64% when cells were treated with 10 ⁇ M of the antisense oligo (ptnASl, SEQ ID N0:1) for 48 hours prior to seeding in soft agar. Analogous treatment with an equivalent concentration of the scrambled control oligo (ptnSCRl, SEQ ID N0:2) , or with the vehicle, did not inhibit the colony forming potential of SW-13/PTN cells.
  • SW-13 cells that had been transfected with an MK expression vector were treated with PTN antisense oligos and assayed the supernatants assayed for colony stimulating activity. MK synthesis also was measured by ELISA. SW-13/MK cells have been shown to form colonies in soft agar due to MK expression. Sale et al . (1994). No inhibition of MK bioactivity or drop in secretion of MK into the SW-13/MK supernatants was detected.
  • MK is closely related to PTN (50% sequence identity) , it appears that our PTN antisense oligos are very specific. From these data, it appears that a specific, dose-dependent inhibition of PTN production and secretion can be achieved in vi tro.
  • 1205-LU melanoma cells constitutively express high levels of PTN and form tumors very aggressively. Hartmann et al . , manuscript in preparation (1994) . These cells were used as a model cell line to determine whether PTN plays a role in melanoma growth. Previously, it has been shown that human melanoma cells express PTN mRNA, whereas human melanocytes do not. Fang et al . (1992). The following studies were conducted to determine whether 1205-LU cells require autocrine-acting PTN for their growth and colony formation or, if they use PTN exclusively for paracrine growth stimulation of surrounding tissues.
  • 1205-LU metastatic melanoma cells do not require PTN to form colonies in soft agar. More specifically, when these cells are treated with antisense oligos that inhibit PTN production, they form the same number of colonies with the same size as those cells treated with both the scrambled oligo and PBS. In addition to the colony formation assay, melanoma cell proliferation was measured for one week during treatment with either antisense oligos or scrambled oligos at concentrations up to 20 ⁇ M. Regardless of oligo treatment or dose, cells proliferated to more than 12 times their original density. This suggests that the oligos themselves were not toxic to the melanoma cells.
  • melanoma cells may stimulate SW-13 as well as endothelial cells.
  • Endothelial cells respond to molecules such as FGFs, PTN and TGF- ⁇
  • SW-13 cells respond to FGFs, PTN, as well as IL-1,24 but not TGF- ⁇ .
  • Conditioned media was collected from each 24 hour antisense treatment interval and assayed for PTN using both (i) a PTN-specific ELISA and (ii) SW-13 and endothelial cell responses.
  • conditioned media from 1205-LU cells treated with antisense oligos was added to SW-13 cells that were seeded in a soft agar.
  • the 24 hour samples did not show any significant inhibition of colony formation.
  • 1205-LU cells were plated in coculture to directly stimulate target huBEC cells. The same results were observed as with conditioned media harvested from 1205-LU cells (data not shown) .
  • the huBEC cells cocultured with ptnASl- and ptnAS3-treated 1205-LU cells showed less proliferation than huBEC cells cocultured with control 1205-LU cells.
  • the endothelial cells cocultured with ptnAS3-treated 1205-LU cells showed an almost 50% inhibition of proliferation in one week compared to the control (PBS-treated) coculture. Endothelial cells from the ptnSCR3 group, however, showed no significant decrease in cell proliferation over the same time period.
  • a corresponding ELISA confirmed the inhibition of PTN protein in the respective 1205-LU conditioned media (data not shown) .
  • Endothelial cells which were not cocultured with 1205-LU cells received no cross-feeding activity and were used to determine background proliferation.
  • melanoma cells were treated with oligos and then injected subcutaneously into athymic nude mice. 1205-LU cells were pretreated for 72 hours as described above. This metastatic melanoma cell line grows into detectable tumors within a few days at the local site of injection. At four days after injection, the tumors were found to be at least 10 mm in diameter, regardless of the pretreatment. This is likely due to the fact that tumors can grow to a certain size without recruiting blood vessels for nutrient support. Folkman et al. J. Biol . Chem. 267: 10931 (1992). Diffusion of oxygen and other nutrients should penetrate and feed these cells during the first few days of growth and tumor formation.
  • the tumor must recruit microvasculature and surrounding stroma in a paracrine fashion for its continued growth support past this early stage. This is evidenced by the average doubling of tumor size in the control (PBS or ptnSCRl pretreated cells) by the end of the first week after injection. In contrast, the antisense (ptnASl) pretreated cells showed only a modest increase of less than 10% growth in this same time period. By nine days post-injection, the control tumors approximately tripled in size, while the antisense pretreated tumors increased in size by less than 25%.
  • NAME BENT, Stephen A.

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Abstract

Des oligonucléotides antisens s'hybridant à des segments de l'ARNm correspondant à l'ADNc pour la pléiotrophine inhibent la synthèse de cette dernière in vitro et in vivo. Sont également décrites des compositions pharmaceutiques contenant ces oligonucléotides comme principes actifs.
PCT/US1995/008781 1994-07-18 1995-07-18 Oligonucleotides antisens de pleiotrophine WO1996002257A1 (fr)

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Cited By (9)

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WO1998040095A1 (fr) * 1997-03-12 1998-09-17 Meiji Milk Products Co., Ltd. Compositions preventives et therapeutiques pour nephropathies et hepatites d'origine medicamenteuse
WO1999053943A2 (fr) * 1998-04-17 1999-10-28 Angiogenix, Incorporated Facteurs angiogeniques therapeutiques et procedes d'utilisation
WO2000035473A3 (fr) * 1998-12-18 2000-11-09 Scios Inc Methodes de detection et d'utilisation de genes exprimes de façon differentielle dans des maladies
EP1108436A1 (fr) * 1998-08-24 2001-06-20 Meiji Milk Products Company Limited Medicament pour le traitement et/ou la prevention de l'arteriosclerose et de la reangiostenose post-actp
EP1121598A1 (fr) * 1998-10-06 2001-08-08 Georgetown University Detection de pleiotrophine
US6709855B1 (en) 1998-12-18 2004-03-23 Scios, Inc. Methods for detection and use of differentially expressed genes in disease states
US6770264B2 (en) 2001-11-13 2004-08-03 Noville, Inc. Chewing gum compositions comprising diglycerol
US7141384B1 (en) 1998-10-06 2006-11-28 Georgetown University Detection of pleiotrophin
CN100386435C (zh) * 2005-12-19 2008-05-07 中国人民解放军军事医学科学院放射与辐射医学研究所 抑制多效生长因子基因表达的小干扰rna及其应用

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 187, Number 2, issued September 1992, LAI et al., "Structure of the Human Heparin-Binding Growth Factor Gene Pleiotrophin", pages 1113-1122. *
BREAST CANCER RESEARCH AND TREATMENT, Volume 31, issued 1994, WELLSTEIN, "Growth Factor Targeted and Conventional Therapy of Breast Cancer", pages 141-146. *
CANCER GENE THERAPY, Volume 1, Number 1, issued 1994, TSENG et al., "Antisense Oligonucleotide Technology in the Development of Cancer Therapeutics", pages 65-71. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 267, Number 36, issued December 1992, FANG et al., "Pleiotrophin Stimulates Fibroblasts and Endothelial and Epithelial Cells and is Expressed in Human Cancer", pages 25889-25897. *
JOURNAL OF BIOLOGICAL CHEMISTY, Volume 269, Number 33, issued August 1994, CZUBAYKO et al., "Ribozyme Targeting Elucidates a Direct Role of Pleiotrophin in Tumor Growth", pages 21358-21363. *
S. CROOKE et al., "Antisense Research and Applications", Published 1993, by CRC PRESS (BOCA RATON), pages 8-35. *
SCIENCE, Volume 261, issued August 1993, STEIN et al., "Antisense Oligonucleotides as Therapeutic Agents - is the Bullet Really Magical?", pages 1004-1011. *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998040095A1 (fr) * 1997-03-12 1998-09-17 Meiji Milk Products Co., Ltd. Compositions preventives et therapeutiques pour nephropathies et hepatites d'origine medicamenteuse
US6572851B2 (en) 1997-03-12 2003-06-03 Takashi Muramatsu Method for suppressing or treating drug-induced nephropathy
WO1999053943A2 (fr) * 1998-04-17 1999-10-28 Angiogenix, Incorporated Facteurs angiogeniques therapeutiques et procedes d'utilisation
WO1999053943A3 (fr) * 1998-04-17 2000-01-20 Angiogenix Inc Facteurs angiogeniques therapeutiques et procedes d'utilisation
EP1108436A1 (fr) * 1998-08-24 2001-06-20 Meiji Milk Products Company Limited Medicament pour le traitement et/ou la prevention de l'arteriosclerose et de la reangiostenose post-actp
EP1108436A4 (fr) * 1998-08-24 2002-05-29 Takashi Muramatsu Medicament pour le traitement et/ou la prevention de l'arteriosclerose et de la reangiostenose post-actp
US7820160B2 (en) 1998-08-24 2010-10-26 Medical Therapies Limited Midkine inhibitory compositions for the treatment of angiostenosis
US7309695B2 (en) 1998-08-24 2007-12-18 Takashi Muramatsu Pharmaceutical compositions for the prevention and treatment of atherosclerosis and restenosis after PTCA
US7141384B1 (en) 1998-10-06 2006-11-28 Georgetown University Detection of pleiotrophin
EP1121598A1 (fr) * 1998-10-06 2001-08-08 Georgetown University Detection de pleiotrophine
WO2000035473A3 (fr) * 1998-12-18 2000-11-09 Scios Inc Methodes de detection et d'utilisation de genes exprimes de façon differentielle dans des maladies
US6709855B1 (en) 1998-12-18 2004-03-23 Scios, Inc. Methods for detection and use of differentially expressed genes in disease states
US6770264B2 (en) 2001-11-13 2004-08-03 Noville, Inc. Chewing gum compositions comprising diglycerol
CN100386435C (zh) * 2005-12-19 2008-05-07 中国人民解放军军事医学科学院放射与辐射医学研究所 抑制多效生长因子基因表达的小干扰rna及其应用

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