WO1999042570A1 - Compositions a base de flip cellulaire utilisees dans le traitement des troubles arteriosclereux - Google Patents

Compositions a base de flip cellulaire utilisees dans le traitement des troubles arteriosclereux Download PDF

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Publication number
WO1999042570A1
WO1999042570A1 PCT/US1999/003558 US9903558W WO9942570A1 WO 1999042570 A1 WO1999042570 A1 WO 1999042570A1 US 9903558 W US9903558 W US 9903558W WO 9942570 A1 WO9942570 A1 WO 9942570A1
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flip
nucleic acid
polypeptide
molecule
cell
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PCT/US1999/003558
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English (en)
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Kenneth Walsh
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St. Elizabeth's Medical Center, Inc.
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Priority to AU28698/99A priority Critical patent/AU2869899A/en
Publication of WO1999042570A1 publication Critical patent/WO1999042570A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to methods and compositions for the treatment of arteriosclerotic disorders, such as the excessive death and damage of endothelial cells associated with atherosclerosis.
  • the methods involve administering a cellular FLIP molecule to inhibit apoptosis in vascular endothelial cells.
  • Arteriosclerosis is a disease that is characterized by a thickening and hardening of regions of an arterial wall.
  • Atherosclerosis is a type of arteriosclerosis that affects the large arteries and is often the basis for coronary artery disease, aortic aneurysm, arterial disease of the lower extremities, and cerebrovascular disease.
  • a particular type of atherosclerosis is transplant arteriosclerosis, which refers to atherosclerosis that occurs in the arteries of transplanted organs, e.g., transplanted hearts and kidneys.
  • Transplant arteriosclerosis is a major cause of death in patients who receive transplanted organs.
  • Atherosclerosis is characterized by the formation of fibrous plaques that contain a large number of smooth muscle cells, macrophages, collagen, extracellular lipid, and necrotic cell debris.
  • the accumulation of material in a fibrous plaque results in narrowing of the blood vessel lumen which, in turn, restricts arterial blood flow.
  • the fibrous plaques become sufficiently large to block blood flow completely, the organs that are supplied by the artery undergo ischemia and necrosis.
  • the accumulation of fibrous plaques also weakens the artery, an event which frequently results in rupture of the intima, aneurysm and hemorrhage.
  • fragments of the fibrous plaque may detach and form arterial cmboli that can precipitate an aortic aneurysm or arterial disease of the lower extremities.
  • the most frequently used methods for treating atherosclerosis include surgical procedures, drug therapies, and combinations of the foregoing.
  • the drug therapies for treating atherosclerosis are designed to prevent or reduce the accumulation of plaque material.
  • drugs such as diuretics, anti-adrenergic agents, vasodilators, angiotensin-converting enzyme inhibitors, renin inhibitors, HMG-CoA reductase inhibitors, and calcium channel antagonists have been used to treat conditions such as hypertension, hyperlipidemia, and hypercholesterolemia, which contribute to the development of atherosclerosis.
  • anti- inflammatory agents have been prescribed for inhibiting transplant arteriosclerosis; however, this approach has met with limited success.
  • Surgical methods for treating atherosclerosis include coronary bypass surgery, atherectomy, laser procedures, ultrasonic procedures, and balloon angioplasty. Such methods involve significant risk (e.g., of infection, death) to the patient and, even if successful, fibrous plaque formation frequently occurs at the site of vascular anastomoses, causing reclusion of the surgically-treated vessel.
  • Injury to the blood vessel wall e.g., resulting from balloon angioplasty, has been shown to disrupt the protective layer of endothelial cells that line the vascular wall, thereby leading to further endothelial cell damage and death.
  • the recruitment of leukocytes at sites of vascular inflammation is a multistep process that involves tethering, rolling, firm adhesion, and the migration of these cells to the subendothelial space.
  • chemoattractant and adhesion molecules that regulate leukocyte recruitment in response to bacterial infection
  • mechanisms that may actively control the transendothelial cell migration relatively little is known about mechanisms that may actively control the transendothelial cell migration.
  • the invention is based, in part, upon Applicant's discoveries relating to the roles played by an endothelial cell intracellular protein in protecting the vascular wall from injury and, thereby, inhibiting leukocyte extravasation and vascular wall inflammation.
  • Fas ligand is expressed on the normal vascular endothelium and that expression of the Fas ligand is down regulated by the inflammatory cytokine, TNF ⁇ .
  • Applicant's further discoveries include: (1) adenovirus-mediated constitutive Fas ligand expression by the endothelium reduces the leukocyte extravasation that is induced by local treatment with TNF ⁇ ; (2) functional Fas ligand-expressing - j - endothelial cells are not sensitive to Fas ligand-induced apoptosis; (3) dysfunctional Fas ligand- expressing endothelial cells (e.g., cells exposed to oxidized lipid) are sensitive to Fas ligand- induced apoptosis; and (4) dysfunctional endothelial cells (e.g., cells exposed to oxidized lipid) exhibit a higher death rate and reduced cellular FLICE-inhibitory protein (FLIP) mRNA levels compared to end
  • FLIP FLICE-inhibitory protein
  • Fas ligand-Fas receptor signaling pathway is a feature of endothelial cell dysfunction in response to injurious agents (e.g., oxidized lipid, mechanical injury, homocysteine, toxins, viruses) and that abnormal FLIP expression plays an important role in the deregulation of the Fas ligand-Fas receptor signaling pathway.
  • injurious agents e.g., oxidized lipid, mechanical injury, homocysteine, toxins, viruses
  • FLIP expression appears to protect the endothelial cells from Fas ligand-mediated apoptosis.
  • a "death effector domain” is found in MORT-1 FADD upstream of the death domain and occurs in duplicate in two caspases: caspase 8 (MACH/FLICE/Mch-5) and caspase 10 (Mch-4/FLICE- 2). Reportedly, the binding of these caspases to MORT-1/FADD through association of their 5 "death effector domain” motifs and consequent activations of these caspases (reportedly by autoproteolytic cleavage), are critical steps in the initiation of apoptosis.
  • the invention is directed to delivering FLIP molecules (nucleic acids, polypeptides) to endothelial cells to inhibit apoptosis that is mediated by the Fas ligand-Fas receptor signalling pathway.
  • FLIP molecules nucleic acids, polypeptides
  • the method for treating an arteriosclerotic condition may be a surgical method or a drug therapy (e.g., gene therapy).
  • a drug therapy e.g., gene therapy
  • the compositions and methods of the invention are useful for replacing existing drug therapies, as well as for improving the effectiveness of existing therapies for treating conditions that are characterized by excessive vascular smooth muscle cell proliferation. In general, such conditions arc diagnosed by detecting the presence of fibrous plaques in the blood vessel walls of the subject.
  • delivery of a FLIP molecule into the arteries of a subject can be accomplished by attaching a FLIP nucleic acid or a FLIP polypeptide to the surface of a balloon catheter, inserting the catheter into the subject until the balloon portion is located at the site of an occlusion (or a predisposition to an occlusion), and inflating the balloon to contact the balloon surface with the vessel wall at the site of the occlusion.
  • the compositions can be targeted to particular sites within a vessel to prevent or - 6 - reduce leukocyte infiltration and smooth muscle cell proliferation at these sites.
  • the FLIP molecule is delivered in combination with a cytokine that promotes endothelial cell proliferation, or a nucleic acid encoding a cytokine that promotes endothelial cell proliferation.
  • a "FLIP molecule” embraces a “FLIP nucleic acid” and a “FLIP polypeptide”.
  • a "FLIP nucleic acid” refers to a nucleic acid molecule which: (1) has the sequence of
  • the preferred FLIP nucleic acid has the sequence of SEQ. ID NO.l (FLIP-long form). Homologs and alleles of a nucleic acid having the sequence of SEQ. ID NO. l also are embraced within the definition of a "FLIP nucleic acid".
  • the FLIP nucleic acids of the invention include nucleic acids which code for the FLIP polypeptide having the sequence of SEQ. ID NO.2 (the polypeptide sequence encoded by SEQ. ID NO. l), but which differ from the sequence of SEQ. ID NO. l in codon sequence due to the degeneracy of the genetic code.
  • FLIP polypeptides are useful for inhibiting Fas-ligand mediated apoptosis and, in particular, are useful for inhibiting apoptosis in vascular endothelial cells.
  • the preferred FLIP polypeptides of the invention have the amino acid sequence of SEQ. ID NO.2 (the sequence for the human FLIP-L polypeptide) or SEQ. ID NO. 4 (the sequence for the human 1 LIP-S polypeptide).
  • the sequences for the murine' FLIP nucleic acid and corresponding polypeptide are provided in GenBank database and are included in the SEQUENCE LISTING.
  • FLIP polypeptides further embrace functionally equivalent fragments, variants, and analogs of SEQ. ID NOs.
  • the invention also embraces proteins and peptides coded for by any of the foregoing nucleic acids.
  • the invention - 8 - embraces proteins and polypeptides which are coded for by unique fragments of the foregoing nucleic acids. Such proteins and polypeptides are useful, for example, as immunogens for generating antibodies to unique epitopes of the FLIP polypeptide.
  • a method for inhibiting (preventing or reducing) Fas ligand-mediated apoptosis of vascular endothelial cells in vivo or in vitro involves contacting an endothelial cell with an isolated FLIP nucleic acid or polypeptide under conditions to permit the introduction into the endothelial cell of a functional FLIP nucleic acid or FLIP polypeptide.
  • a FLIP nucleic acid contained in an expression vector can be introduced into the cell, followed by allowing the cell to transcribe and translate the FLIP nucleic acid into a FLIP polypeptide in the endothelial cell.
  • a FLIP polypeptide contained in a liposome can be introduced into the cell.
  • the FLIP molecule is introduced into the cell in an amount sufficient to inhibit Fas ligand-mediated apoptosis of the endothelial cell in vivo or in vitro.
  • the FLIP molecule is introduced into the cell in an amount sufficient to inhibit apoptosis that is mediated by contacting the cell with an oxidized lipid (e.g., oxidized LDL).
  • an oxidized lipid e.g., oxidized LDL
  • an isolated FLIP nucleic acid is administered to a subject in need thereof in an amount effective to inhibit (prevent or reduce) Fas ligand-mediated apoptosis of vascular endothelial cells in vivo.
  • the subjects are treated with the FLIP nucleic acid in a manner and in an amount so as to inhibit vascular endothelial cell apoptosis, preferably at a site of vascular wall inflammation or a predisposition to vascular wall inflammation, while minimizing the potential for systemic toxicity.
  • Further specificity of treatment is achieved by operably coupling the FLIP nucleic acid to an inducible promoter or a tissue-specific promoter, such as an endothelial cell-specific promoter.
  • the promoter is one which is not down regulated by oxidized lipid (e.g., strong viral promoters such as CMV).
  • an isolated FLIP polypeptide is administered to a subject in need thereof in an amount effective to inhibit Fas ligand-mediated apoptosis of vascular endothelial cells in vivo ' .
  • the subjects are treated with the FLIP polypeptide in a manner and in an amount so as to inhibit vascular endothelial cell apoptosis, preferably at the site of vascular wall inflammation or a predisposition to vascular wall inflammation, while minimizing the potential for systemic toxicity.
  • compositions and/or methods use these and other mammalian FLIP isoforms as provided in the GenBank database (see also Sequence
  • the FLIP nucleic acid has the nucleotide sequence of SEQ. ID NO 1 (human FLIP-L), i.e., the complete coding sequence of the mRNA encoding the "intact human FLIP-L".
  • the intact human FLIP-L polypeptide contains two "death effector domains" (DED I and DED II) and a caspase-like domain containing two caspase-like subunits ("pi 7 subuni ', "pi 2 subunif) that have homology to the pi 7 and the pi 2 subunits, respectively, of FLICE.
  • DED I and DED II two "death effector domains”
  • caspase-like domain containing two caspase-like subunits
  • pi 7 subuni ', "pi 2 subunif caspase-like subunits
  • DED I contains amino acids 1 to 74
  • DED II contains amino acids 93 to 171
  • homology to the pi 7 FLICE subunit is from amino acid 242 to 376 in FLIP-L
  • homology to the pi 2 FLICE subunit is from amino acid 377 to.480 in FLIP-L
  • the numbering is based upon that reported by M. Irmler, et al, in Nature 388:190-195 (1997) for the human cellular FLIP.
  • sequence numbers that are provided are intended to be inclusive, e.g., DED I contains amino acids 1 to 74, inclusive.
  • the isolated nucleic acids of the invention also include nucleic acids encoding fragments of an intact FLIP.
  • the FLIP nucleic acid may encode one or more DED domains, alone or in combination with one or more caspase-like subunits.
  • exemplary FLIP nucleic acids include those which encode: DED L DED II, DED I coupled to DED II, multiples of the - 10 - foregoing DED domains, alone or in combination with one or more of the caspase-like subunits pl7 and pl2.
  • the FLIP nucleic acids encode DED I (amino acids 1-74), DED II (amino acids 93-171), and DED I coupled to DED II (e.g., amino acids 1-74 coupled directly to amino acids 93-171 of SEQ. ID NO. 1, amino acids 1-171 of SEQ. ID NO. 1).
  • DED I amino acids 1-74
  • DED II amino acids 93-171
  • DED I coupled to DED II e.g., amino acids 1-74 coupled directly to amino acids 93-171 of SEQ. ID NO. 1, amino acids 1-171 of SEQ. ID NO. 1).
  • the FLIP nucleic acids further contain one or more caspase-like subunits.
  • FLIP polypeptide fragments that are "FRAGMENT 1" contain DED I and DED II (e.g., amino acids 1 to 171, with or without intervening amino acids 75-92 of FLIP-L, SEQ. ID NO.2) but do not include one or more of caspase-like domains (e.g., amino acids 172 to the carboxyl- terminus of SEQ. ID NO.2, as numbered by Irmler et al., Nature, supra.). FLIP FRAGMENT 1 polypeptides previously have not been described.
  • DED I and DED II e.g., amino acids 1 to 171, with or without intervening amino acids 75-92 of FLIP-L, SEQ. ID NO.2
  • caspase-like domains e.g., amino acids 172 to the carboxyl- terminus of SEQ. ID NO.2, as numbered by Irmler et al., Nature, supra.
  • one particular aspect of the invention relates to such FLIP FRAGMENT 1 polypeptides, nucleic acids encoding same, complements of said nucleic acids, vectors containing said nucleic acids, host cells containing said vectors, antibodies that selectively bind to said polypeptides but that do not bind to the intact FLIP polypeptides, and methods for using the foregoing compositions.
  • the invention further embraces nucleic acid molecules that differ from the foregoing isolated nucleic acid molecules in codon sequence due to the degeneracy of the genetic code. Throughout this document, it is intended that the cDNAs corresponding to the mRNAs that are provided in the sequences also are embraced within the meaning of the phrase, "FLIP nucleic acid”.
  • the FLIP nucleic acid is selected from the group consisting of an intact FLIP-L nucleic acid (e.g., SEQ. ID NO.l, the coding region of SEQ. ID NO.l), a FLIP FRAGMENT 1 containing DED 1 and DED II (e.g., the nucleic acid sequence of SEQ. ID NO. 1 that encodes amino acids 1 to 171 of SEQ. ID NO. 2), a FLIP FRAGMENT 2 containing DED I but excluding DED II (e.g., the nucleic acid sequence of SEQ. ID NO. 1 that encodes amino acids 1 to 74 of SEQ. ID NO.
  • an intact FLIP-L nucleic acid e.g., SEQ. ID NO.l, the coding region of SEQ. ID NO.l
  • a FLIP FRAGMENT 1 containing DED 1 and DED II e.g., the nucleic acid sequence of SEQ. ID NO. 1 that encodes amino acids 1 to 171 of SEQ
  • a FLIP FRAGMENT 3 containing DED II but excluding DED I e.g., the nucleic acid sequence of SEQ. ID NO. 1 that encodes amino acids 93 to 171 of SEQ. ID NO. 2
  • DED I e.g., the nucleic acid sequence of SEQ. ID NO. 1 that encodes amino acids 93 to 171 of SEQ. ID NO. 2
  • caspase-like subunits e.g., the nucleic acid sequence of SEQ. ID NO. 1 that encodes amino acids 242 to 376 and/or amino acids 377 to 480 of SEQ. ID NO. 2
  • the preferred FLIP nucleic acid fragments encode a functionally equivalent fragment of an intact FLIP.
  • the FLIP nucleic acid is operatively coupled to a promoter that can express the - 11 -
  • the FLIP in a targeted cell e.g., a vascular endothelial cell
  • the nucleic acid is contained in an appropriate expression vector (e.g., adenoviral vector, modified adenoviral vector, retroviral vector, plasmid, liposome) to more efficiently genetically modify the targeted cell and achieve expression of the FLIP on the targeted cell surface.
  • an appropriate expression vector e.g., adenoviral vector, modified adenoviral vector, retroviral vector, plasmid, liposome
  • the FLIP nucleic acid promoter is one which is not down regulated, directly or indirectly, by oxidized lipid, e.g., the CMV promoter.
  • vascular endothelial cells express the Fas receptor and that such cells become susceptible to Fas ligand-mediated apoptosis under conditions of reduced FLIP expression (e.g., conditions associated with elevated vascular oxidized lipid levels).
  • delivery of a nucleic acid encoding the FLIP polypeptide to a site of vascular wall inflammation (or a predisposition to vascular wall inflammation) serves to inhibit Fas ligand-mediated apoptosis of vascular endothelial cells, thereby promoting the beneficial effects of viable endothelial cells in the vessel wall.
  • a method for treating vascular wall inflammation in a subject is provided.
  • a FLIP nucleic acid is administered to a subject in need of such treatment in an amount effective to inhibit Fas ligand-mediated apoptosis of vascular endothelial cells.
  • the preferred FLIP nucleic acids are as described herein.
  • the FLIP molecules are particularly useful for the treatment of atherosclerosis and transplant arteriosclerosis.
  • the effective amount is sufficient to inhibit Fas ligand-mediated apoptosis of vascular endothelial cells in vivo.
  • the above FLIP molecules (FLIP nucleic acids and FLIP polypeptides) are used in the preparation of medicaments, preferably for the treatment of conditions characterized by vascular wall inflammation. Such conditions include atherosclerosis and transplant arteriosclerosis.
  • Atherosclerosis is associated with the following conditions, each of which can be treated using the compositions of the invention: restenosis, o pulmonary hypertension, and vascular remodeling.
  • the method involves placing the FLIP molecules in a pharmaceutically-acceptable carrier.
  • the preferred FLIP molecules are as described herein.
  • the invention also contemplates the use of FLIP molecules in experimental model systems to determine the role that vascular endothelial cells play in the repair of an injury to a vessel wall or in mediating an adverse health consequence occurring as a result of organ transplant.
  • a blood vessel of an animal or a pulmonary hypertensive state is induced o experimentally, for example, by hypercholesterolemic diet and/or by inducing a hypoxic state at a particular site.
  • a FLIP molecule as described above then is administered to the animal.
  • the application may be local or may be systemic. Then the animal's response is monitored and compared to control animals that do not receive the FLIP molecules.
  • organ transplant can be performed in an animal model, with or without treatment of the organ and/or animal with 5 a FLIP molecule.
  • the application of the FLIP, molecule may be local or may be systemic. Then the animal's response is monitored and compared to control animals (and/or control organs) that do not receive the FLIP molecules to assess the effectiveness of FLIP in inhibiting Fas ligand- mediated apoptosis of vascular endothelial cells in vivo.
  • a screening method for selecting an o inhibitory agent that inhibits the development or progression of atherosclerotic legions involves: (1) contacting a cell that expresses FLIP with an oxidized lipid and a putative inhibitor under conditions wherein the cell is capable of undergoing Fas ligand- - 13 - induced apoptosis; (2) determining whether the cell undergoes Fas ligand-induced apoptosis in the presence of the putative inhibitor; and (3) selecting the putative inhibitor that inhibits or prevents Fas ligand-induced apoptosis as an agent that inhibits the development or progression or atherosclerotic legions.
  • the putative inhibitor is a relatively small synthetic compound and, more preferably, is contained in a combinatorial or peptidc library that contains a mixture of at least ten different compounds.
  • a kit includes: (1) an oxidized lipid (e.g., oxidized LDL); (2) a cell that expresses FLIP and that is capable of undergoing Fas ligand-induced apoptosis when FLIP expression is down regulated (e.g., an endothelial cell); (3) a Fas ligand or a Fas ligand mimic (e.g., antibody that binds to Fas receptor and induces apoptosis); and (4) instructions for inducing apoptosis and determining whether a test compound inhibits or prevents Fas ligand-induced apoptosis.
  • an oxidized lipid e.g., oxidized LDL
  • a cell that expresses FLIP and that is capable of undergoing Fas ligand-induced apoptosis when FLIP expression is down regulated e.g., an endothelial cell
  • a Fas ligand or a Fas ligand mimic e.g.,
  • FIG. 2A is a series of graphs illustrating that OxLDL induces DNA fragmentation.
  • HUVECs (70% confluent) were incubated in the presence or absence of OxLDL (300 ⁇ g protein/ml), a neutralizing anti-FasL antibody (10 ⁇ g/ml, 4H9) or an agonistic anti-Fas antibody (0.5 ⁇ g/ml, CHI 1 , MBL) for 16 hours in combinations as indicated.
  • Hamster IgG was used as an isotype-matched control for 4H9.
  • Floating and attached cells were harvested by trypsinization, fixed in 4% paraformaldehyde, pcrmeabilizcd in 0.1 % Triton X- 100, 0.1 % sodium citrates, and - 14 - incubated with TdT-mediated dUTP nick end labeling (TUNEL) solution (Boehringer Mannheim) in the absence (open curves) and in the presence (semi-filled curves) of terminal deoxynucleotidyl transferase. Fluorescence intensity was analyzed by FACS.
  • TUNEL TdT-mediated dUTP nick end labeling
  • Figure 2B is a bar graph illustrating that OxLDL impairs cell viability.
  • HAECs were cultured in a 96-well plate at 80% eonfluency and treated with OxLDL (300 ⁇ g protein/ml), a neutralizing anti-FasL antibody (10 ⁇ g/ml, 4H9) and an anti-Fas antibody (0.5 ⁇ g/ml, CHI 1) for 18 hours in combinations as indicated.
  • Cell viability was determined by means of MTT (Dimethylthiazol-diphenyltetrazolium bromide) assay (Tanaka et al., 1996). Data are presented as mean ⁇ S.E.M.
  • Figure 3 is a series of graphs illustrating the dose-response and time course relationships for lipid-induced reduction in HUVEC viability. MTT assays were performed to determine cell viability. Dose-response curves were performed on HUVECs after 12 hours incubation with OxLDL (Fig. 3A) or LPC (Fig. 3B, 3C). Time course measurements were made with 70 ⁇ M LPC.
  • Figure 4 is a schematic representation illustrating the effects of LBC on the expression of various death ligand/death receptor pathway components. Ribonuclease protection assays were performed to determine the effects of LPC on the indicated mRNA transcripts.
  • HUVECS were treated with LPC (70 ⁇ M) for the indicated times prior to RNA extraction. LPC had little or no effect on the expression of many of the cell death pathway components.
  • Figure 5 is a schematic representation illustrating that LPC downregulates FLIP and upregulates FasL transcript expression. HUVECS were treated with LPC (70 ⁇ M) for the indicated times prior to RNA extraction. Northern blot analysis was performed using FLIP or FasL cDNA probes. Two FLIP transcripts were detected: FLIP L (upper) and FLIP S (lower). The 28S ribosome band is shown to indicate equal loading.
  • FIG. 7 schematically illustrates a representative Adeno-FLIP construct according to the invention.
  • SEQ ID NO: 1 is the nucleic acid encoding human FLIP-L and having GenBank Accession No. U97074.
  • SEQ ID NO:2 is the human FLIP-L polypeptide encoded by SEQ. ID NO. 1 and having GenBank Accession No. U97074.
  • SEQ ID NO:3 is the nucleic acid encoding human FLIP-S and having GenBank Accession No. U97075.
  • SEQ ID NO:4 is the human FLIP-S polypeptide encoded by SEQ. ID NO. 3 and having
  • SEQ ID NO:5 is the nucleic acid encoding murine FLIP-L and having GenBank Accession No. U97076.
  • SEQ ID NO:6 is the murine FLIP-L polypeptide encoded by SEQ. ID NO. 5 and having GenBank Accession No. U97076.
  • SEQ ID NOJ is the nucleic acid encoding an alternative spliced form of human FL ⁇ ME- 1-delta and having GenBank Accession No. AF009619.
  • SEQ ID NO:8 is the alternative spliced form of human FLAME-1-delta polypeptide encoded by SEQ. ID NO. 7 and having GenBank Accession No.AF009619.
  • SEQ ID NO:9 is the nucleic acid encoding human FLAME-1 and having GenBank
  • SEQ ID NO: 10 is the human FLAME-1 polypeptide encoded by SEQ. ID NO. 9 and having GenBank Accession No.AF009616.
  • SEQ ID NO: 11 is the nucleic acid encoding an alternative spliced form of human FLAME-1 -beta and having GenBank Accession No. AF009617.
  • SEQ ID NO: 12 is the alternative spliced form of human FLAME-1 -beta polypeptide encoded by SEQ. ID NO. 11 and having GenBank Accession No.AF009617. of viability.
  • These assays revealed greater ⁇ -gal-positive cells in cultures co-transfected with FLIP or Bcl-X L expression vectors than with the control vector (pCDNA) or mock transfected (TE). This increase in the number of surviving ⁇ -gal-positive cells indicates that plasmid- medicated FLIP expression can reverse the apoptosis caused by LPC, a component of OxLDL.
  • Figure 7 schematically illustrates a representative Adeno-FLIP construct according to the invention.
  • SEQ ID NO:6 is the murine FLIP-L polypeptide encoded by SEQ. ID NO. 5 and having o GenBank Accession No. U97076.
  • SEQ ID NOJ is the nucleic acid encoding an alternative spliced form of human FL ⁇ ME- 1-delta and having GenBank Accession No. AF009619.
  • SEQ ID NO: 11 is the nucleic acid encoding an alternative spliced form of human o FLAME-1-beta and having GenBank Accession No. AF009617.
  • the invention is based on the discovery that vascular endothelial cells express the Fas receptor but that cellular FLIP protects the endothelial cells from Fas ligand-mediated apoptosis.
  • Endothelial cells normally express the Fas receptor but are immune to Fas ligand-mediated apoptosis. Based upon these observations and the experimental results described herein, it is believed that FLIP's physiological function in endothelial cells is to protect these cells from Fas ligand-mediated apoptosis. Applicant has further discovered that under conditions of elevated oxidized lipid, endothelial cells become susceptible to Fas ligand-mediated apoptosis and that such susceptible cells exhibit reduced FLIP transcription. Although not wishing to be bound to one particular theory or mechanism, Applicant believes that these observations form the foundation for novel therapeutic methods and related compositions for treating conditions that arc characterized by vascular endothelial cell death and vascular wall inflammation.
  • the invention involves the use of a nucleic acid encoding FLIP ("FLIP nucleic acid”) to express one or more copies of the FLIP polypeptide in endothelial cells and, in particular, to express the FLIP polypeptide in endothelial cells that are susceptible to Fas ligand- mediated apoptosis.
  • FLIP nucleic acid a nucleic acid encoding FLIP
  • infection of the vessel wall with, for example, a FLIP nucleic acid- containing vector (e.g., viral vector, plasmid), results in expression of FLIP polypeptide in the endothelial cells, thereby rendering the cells resistant to the Fas ligand-mediated apoptosis.
  • a FLIP nucleic acid- containing vector e.g., viral vector, plasmid
  • GenBank Accession No. U97074 (SEQ. ID NOS. 1 and 2) for the human cellular FLIP-L mRNA and - 18 - predicted amino acid sequences, respectively; GenBank Accession No. U97075 (SEQ. ID NOS. 3 and 4) for the human cellular FLIP-S mRNA and predicted amino acid sequences, respectively; and GenBank Accession No.U97076 (SEQ. ID NOS. 5 and 6) for the murine cellular FLIP-L mRNA and predicted amino acid sequences, respectively.
  • the invention is directed to a method for treating a subject diagnosed as having a condition characterized by vascular wall inflammation and, optionally, lurther characterized by elevated vascular levels of oxidized lipids.
  • vascular wall inflammation characterized by vascular wall inflammation and, optionally, lurther characterized by elevated vascular levels of oxidized lipids.
  • vascular wall inflammation include, but are not limited to, the following diseases: arteriosclerosis, including atherosclerosis, transplant arteriosclerosis; post interventional restenosis or other vessel wall injury-induced excessive vascular smooth muscle cell proliferation resulting from endothelial cell dysfunction.
  • the method involves administering to the subject an isolated FLIP molecule in an amount and in a manner effective to inhibit (prevent or reduce the progression of) Fas ligand-mediated apoptosis of vascular endothelial cells in vivo.
  • the FLIP molecules may, alternatively, be administered by perfusing or soaking the organ in a solution containing the FLIP molecules prior to implantation.
  • One aspect of the invention involves the use of the FLIP molecules of the invention for WO 9 y 9 y / / 4 4 2 ⁇ 57 / 0 ⁇ PCT/US99/03558
  • the most widely used method to achieve revascularization of a coronary artery is percutaneous transluminal coronary angioplasty.
  • a flexible guide wire is advanced into a coronary artery and positioned across the stenosis.
  • a balloon catheter then is advanced over the guide wire until the balloon is positioned across the stenosis.
  • the balloon then is repeatedly inflated until the stenosis is substantially eliminated.
  • This procedure as compared to heart surgery, is relatively noninvasive and typically involves a hospital stay of only a few days. The procedure is an important tool in the management of serious heart conditions.
  • Pulmonary hypertension as used herein means a right ventricular systolic or a pulmonary artery systolic pressure, at rest, of at least 20 mmHg. Pulmonary hypertension is measured using conventional procedures well-known to those of ordinary skill in the art. Pulmonary hypertension can have a variety of etiologies. - 20 -
  • isolated means a nucleic acid sequence: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) synthesized by, for example, chemical synthesis; (iii) recombinantly produced by cloning; or (iv) purified, as by cleavage and gel separation.
  • isolated as used herein in reference to a polypeptide (protein), means a polypeptide encoded by an isolated nucleic acid sequence, as well as polypeptides synthesized by, for example, chemical synthetic methods, and polypeptides separated from biological materials, and then purified using conventional protein analytical procedures.
  • the FLIP nucleic acid has the nucleotide sequence of SEQ. ID NO 1, the nucleotide sequence encoding an "intact FLIP polypeptide", i.e., the complete coding sequence of the gene encoding human FLIP-L.
  • the isolated FLIP nucleic acids of the invention also include nucleic acids encoding fragments of an intact FLIP.
  • the fragments are functional equivalents of the intact FLIP nucleic acid.
  • the FLIP nucleic acids may encode a fragment that contains one or more DED sequences, alone or coupled to one another and, optionally, further in combination with one or more caspase-like subunits as described above.
  • the FLIP nucleic acid is selected from the group consisting of an intact FLIP nucleic acid (e.g., SEQ. ID NO.l , SEQ. ID NO. 3), and any fragments of SEQ. ID Nos. 1 or 3 which contain one or more DED sequences, alone or coupled to one or more caspase-like subunits as described herein.
  • the FLIP nucleic acid is operatively coupled to a promoter that can express the FLIP polypeptide in a targeted cell (e.g., a vascular endothelial cell). More preferably, the FLIP nucleic acid is operatively coupled to a promoter that is not down regulated, directly or indirectly, by oxidized lipid.
  • promoters that can be used for this purpose include strong viral promoters such as CMV.
  • the nucleic acid is contained in an appropriate expression vector (e.g., adenoviral vector, modified adenoviral vector, retroviral vector, plasmid, liposome) to more efficiently genetically modify the targeted cell and achieve expression of multiple copies of the FLIP polypeptide in the target cell.
  • an appropriate expression vector e.g., adenoviral vector, modified adenoviral vector, retroviral vector, plasmid, liposome
  • the FLIP nucleic acids of the invention can be identified by conventional techniques, e.g., by identifying nucleic acid sequences which code for FLIP polypeptides and which have the sequence of SEQ. ID Nos. 1 or 3 or which hybridize to a nucleic acid molecule having the sequence of SEQ. ID NOs.l or 3 under stringent conditions.
  • stringent conditions refers to parameters with which the art is familiar.
  • stringent conditions refer to hybridization at 65 °C in hybridization buffer (3.5 x SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02% bovine serum albumin, 2.5mM NaH 2 P0 4 (pH 7), 0.5% SDS, 2mM EDTA).
  • SSC is 0.15M sodium chloride/0.15M sodium citrate, pH 7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetraacetic acid.
  • the membrane to which the DNA is transferred is washed at 2x SSC at room temperature and then at 0.1x SSC/0.1x SDS at 65°C.
  • homologs and alleles typically will share at least 40% nucleotide identity with SEQ. ID. NOs. 1 or 3, or a cDNA that is transcribed into SEQ. ID Nos. 1 or 3; in some instances, will share at least 50% nucleotide identity; and in still other instances, will share at least 60% nucleotide identity.
  • Watson-Crick complements of the foregoing nucleic acids are also embraced by the invention.
  • the preferred homologs have at least 70% sequence homology to SEQ. ID.
  • the invention also includes degenerate nucleic acids which include alternative codons to those present in the naturally occurring nucleic acid that codes for the human FLIP polypeptide.
  • serine residues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC.
  • each of the six codons is equivalent for the purposes of encoding a serine residue.
  • any of the serine-encoding nucleotide codons may be employed to direct the protein synthesis apparatus, in vitro or in vivo, to incorporate a serine residue.
  • nucleotide sequence triplets which encode other amino acid residues include, but are not limited to, CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG,
  • CGT, AGA and AGG arginine codons
  • ACA ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons).
  • Other amino acid residues may be encoded similarly by multiple nucleotide sequences.
  • the invention embraces degenerate nucleic acids that differ from the naturally occurring isolated nucleic acids in codon sequence due to the degeneracy of the genetic code.
  • the invention also provides isolated unique fragments of SEQ. ID NO. l and SEQ. ID NO. 2, and complements of the foregoing FLIP nucleic acids.
  • a unique fragment is one that is a - 24 -
  • Unique fragments can be used as probes in Southern blot assays to identify family members or can be used in amplification assays such as those employing PCR. As known to those skilled in the art, large probes such as 200 base pair (BP) or more are preferred for certain uses such as Southern blots, while smaller fragments will be preferred for uses such as PCR. The fragments are also useful as probes for mRNA in Northern blot analysis. Unique fragments also can be used to produce fusion proteins for generating antibodies or for generating immunoassay components.
  • Unique fragments are also useful for a variety of assays to determine the protein binding regions of the nucleic acid, such as gel shift assays and can be cloned into reporter constructs such as a chloramphenicol acetyl transferase (CAT) vector to determine the active promoter and enhancer regions.
  • reporter constructs such as a chloramphenicol acetyl transferase (CAT) vector to determine the active promoter and enhancer regions.
  • unique fragments can be employed to produce fragments of the FLIP polypeptide, such as a "FRAGMENT 1 " (described below) FLIP polypeptide, useful, for example, in inhibiting Fas ligand-mediated apoptosis in Fas ligand receptor-expressing cells (e.g., endothelial cells) that contact a Fas ligand or a Fas ligand-expressing cell.
  • Complements of unique fragments further can be used as antisense molecules to inhibit the expression of the FLIP polypeptide, particularly
  • the size of the unique fragment will depend upon its conservancy in the genetic code. Thus, some regions of SEQ. ID. Nos. 1 or 3, will require longer segments to be unique while others will require only short segments, typically between 12 and 32 base pairs. Virtually any segment of SEQ. ID Nos.1 or 3, that is 1 or more nucleotides in length will be unique. Those skilled in the art are well versed in methods for selecting such sequences, typically on the basis of the ability of the unique fragment to selectively distinguish the sequence of interest from other family members. Unique fragments of the FLIP polypeptides of the invention, nucleic acids encoding same, are a particularly preferred aspect of the invention.
  • the FLIP nucleic acid in one embodiment, is operably linked to a gene expression sequence which directs the expression of the FLIP nucleic acid within a eukaryotic cell.
  • gene expression sequence is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enliancer combination, which facilitates the efficient transcription and translation of the FLIP nucleic acid to which it is operably linked.
  • the gene expression sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter.
  • Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate kinase, ⁇ -actin promoter and other constitutive promoters.
  • Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the simian virus, papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus (CMV), the long terminal repeats (LTR) of moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus.
  • Other constitutive promoters are known to those of ordinary skill in the art.
  • the promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are modulated in the presence of an inducing agent.
  • the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions.
  • Other inducible promoters are known to those of ordinary skill in the art and include the tetracycline- based, high-level gene expression systems described by Bujard, Gossen, and colleagues (Gossen, M. & Bujard, H., 1992, Proc. Nail. Acad. Sci. USA, 89:5547-5551; Gossen, M. et al., 1995, Science, 268: 1766-1769.
  • the preferred promoters for use in connection with the instant invention are those that are not down-regulated (directly or indirectly) by oxidized lipid.
  • the gene expression sequence shall include, as necessary, 5' non-transcribing and 5' non-translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CA ⁇ T sequence, and the like.
  • 5' non-transcribing sequences will include a promoter region which includes a promoter sequence for transcriptional control of the ⁇ operably joined FLIP nucleic acid.
  • the gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired.
  • the FLIP nucleic acid of the invention is linked to a gene expression sequence which permits expression of the FLIP nucleic acid in an endothelial cell. More preferably, the gene expression sequence permits expression of the FLIP nucleic acid in a human vascular endothelial cell and does not permit expression of the FLIP nucleic acid in smooth muscle cells, hepatocytes and other Fas receptor-expressing cell types because it is undesirable to interfere with the normal apoptosis of these cells.
  • a sequence which permits expression of the FLIP nucle i c acid in a human vascular endothelial cell is one which is selectively active in vascular endothelial cells and thereby causes the expression of the FLIP nucleic acid in these cells.
  • the following promoters can be used to express the FLIP nucleic acid in human vascular endothelial cells: CMV, Tie2 gene promoter.
  • CMV CMV
  • Tie2 gene promoter Those of ordinary skill in the art will be able to easily identify alternative promoters that are capable of expressing a FLIP nucleic acid in a vascular endothelial cell.
  • the FLIP nucleic acid sequence and the gene expression sequence are said to be "operably linked” when they are covalently linked in such a way as to place the transcription and/or translation of the FLIP coding sequence under the influence or control of the gene expression sequence.
  • two DNA sequences are said to be operably linked if induction of a promoter in the 5' gene expression sequence results in the transcription of the FLIP sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the FLIP sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a gene expression sequence would be operably linked to a FLIP nucleic acid sequence if the gene expression sequence were capable of effecting transcription of that FLIP nucleic acid sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
  • the FLIP nucleic acids of the invention can be delivered to the vascular endothelial cell alone or in association with a vector.
  • a "vector” is any vehicle capable of facilitating: (1) delivery of a FLIP molecule to a target cell and/or (2) uptake of a FLIP molecule by a target cell.
  • the vectors transport the FLIP molecule into the target cell with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • a "targeting ligand" can be attached to the vector to selectively deliver the vector to a cell which expresses on its surface the cognate receptor for the targeting ligand.
  • the vector (containing a FLIP nucleic acid or a FLIP protein) can be selectively delivered to a vascular endothelial cell in, e.g., the arterial wall.
  • Methodologies for targeting include conjugates, such as those described in U.S. Patent 5,391 ,723 to Priest.
  • Another example of a well-known targeting vehicle is a liposome. Liposomes are commercially available from Gibco BRL. Numerous methods are published for making targeted liposomes.
  • FLIP molecules of the invention are targeted for delivery to an endothelial cell and. more preferably, a vascular endothelial cell.
  • the vectors useful in the invention are divided into two classes: biological vectors and chemical/physical vectors.
  • biological vectors are useful for delivery /uptake of FLIP nucleic acids to/by a target cell.
  • Chemical/physical vectors are useful for delivery /uptake of FLIP nucleic acids or FLIP proteins to/by a target cell.
  • Bio vectors include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the nucleic acid sequences of the invention, and additional nucleic acid fragments (e.g., enhancers, promoters) which can be attached to the nucleic acid sequences of the invention.
  • additional nucleic acid fragments e.g., enhancers, promoters
  • Viral vectors are a preferred type of biological vector and include, but are not limited to, nucleic acid sequences from the following viruses: adenovirus; adeno-associated virus; retrovirus, such as moloney murine leukemia virus; harvey murine sarcoma virus: murine mammary tumor virus; rouse sarcoma virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • viruses include, but are not limited to, nucleic acid sequences from the following viruses: adenovirus; adeno-associated virus; retrovirus, such as moloney murine leukemia virus; harvey murine sarcoma virus: murine mammary tumor virus; rouse sarcoma virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papill
  • a particularly preferred virus for certain applications is the adeno-associated virus, a double-stranded DNA virus.
  • the adeno-associaled virus is capable of infecting a wide range of cell types and species and can be engineered to be replication-deficient. It further has advantages, such as heat and lipid solvent stability, high transduction frequencies in cells of diverse lineages, including hemopoietic cells, and lack of superinfection inhibition thus allowing multiple series of transductions.
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression.
  • adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • the preparation of an adeno-associated virus containing a nucleic acid encoding the intact human FLIP is described in the Examples.
  • This construct is designated "Adeno-FLIP" and contains a serotype 5 human replication defective adenovirus encoding the full-length human FLIP polypeptide from the CMV promoter/enhancer).
  • Ad-FLIP contains a serotype 5 human replication defective adenovirus encoding the full-length human FLIP polypeptide from the CMV promoter/enhancer.
  • other isoforms of the FLIP polypeptide coding sequences can be substituted for that described in the Examples to provide alternative constructs - 28 - that are useful for practicing the inventions disclosed herein.
  • Adeno-FLIP constructs can be constructed by subcloning a human FLIP cDNA, e.g., based upon the sequences for FLIP-L (Accession # U97074) or FLIP-S (Accession # U97075). downstream from an appropriate expression cassette (for example, the CMV promoter/enhancer) into the EcoRV site of the pCOl vector containing the Ad5 adenoviral sequences required for homologous recombination. The resulting plasmid can then be linearized by restriction enzyme digestion and cotransfected in 293 cells with large Clal fragment of the Ad5 dl324 viral DNA (Stratford-Perricaudet, L.D., et al., 1993, J. Clin.
  • the resulting replication- defective recombinant adenoviral constructs are then purified from isolated plaques.
  • the viral preparations are typically purified by two CsCl gradient centrifugations, dialyzed against buffer containing 10 mM Tris-Cl pH 7.5, 1 mM MgCl 2 and 135 mM NaCl and stored at -80 °C in 10% glycerol.
  • Viral titer is typically determined by plaque assay on 293 cells (Graham, F.L., and A.J. van der Eb, 1973, Virology 52:456-463) and expressed as plaque forming units (pfu) per ml.
  • adeno-FLIP constructs can be constructed by substituting an inducible promoter (e-g-, a tetracycline-based cassette) for the constitutive promoter (e.g. the CMV promoter/enhancer).
  • an inducible promoter e.g. the CMV promoter/enhancer.
  • Preferred tetracycline-based inducible promoter systems include the Tet-OffTM & Tet-OnTM Gene Expression Systems available from Clontech, Palo Alto, CA. Briefly, Tet-Off and Tet-On Gene Expression Systems allow high-level, regulated gene expression in response to varying concentrations of tetracycline (Tc) or Tc derivatives such as doxyeycline (Dox). In the Tet-Off System, gene expression is turned on in the absence of Tc or
  • Tet Expression Systems are based on two regulatory elements derived from the tetracycline-resistance operon of the E. coli Tnl O transposon ⁇ the tetracycline repressor protein (TetR) and the tetracycline operator sequence (tetO) to which TetR binds.
  • TetR tetracycline repressor protein
  • tetO tetracycline operator sequence
  • the gene to be expressed (e.g., FLIP-L: SEQ ID NO: 1, FLIP-S: SEQ ID NO:3, FLIP-homologs, etc.) is cloned into the pTRE "response" plasmid, which contains the PhCMV*-l promoter upstream of a multiple cloning site (MCS).
  • PhCMV*-l is a compound promoter consisting of the tetracycline -responsive element (TRE), which contains seven copies of tetO, and the minimal immediate early promoter of cytomegalovirus (PminCMV).
  • the second key component of the system is a "regulator" plasmid which expresses a hybrid protein known as the Tc-controlled transactivator (tTA).
  • tTA is encoded by pTet-Off and is a fusion of the wild-type TetR to the VP16 activation domain (AD) of herpes simplex virus.
  • tTA binds the tetO sequences which - 29 - brings the VP16 activation domain into close proximity with the PhCMV*-l --and thereby activates transcription—in the absence of Tc.
  • Tc is added to the culture medium, transcription is turned off in a dose-dependent manner.
  • the Tet-On System is based on the "reverse" TetR (rTetR), which differs from the wild-type TetR by four amino acid changes (Gossen, M, et al. , 1995, Science, 268:1766-1769). When fused to the VP16 AD, rTetR creates a "reverse" tTA (rtTA) that activates transcription in the presence of Tc or Dox. As described in the examples, the Tet-On System was utilized to generate the tetracycline-responsive FLIP adenoviral constructs.
  • Non-cytopathic viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest.
  • Non-cytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • Adenoviruses and retroviruses have been approved for human gene therapy trials.
  • the retroviruses are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle).
  • retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • Another preferred retroviral vector is the vector derived from the moloney murine leukemia virus, as described i Nabel, E.G., et al., Science, v. 249, p. 1285-1288 (1990). These vectors reportedly were effective for the delivery of genes to all three layers of the arterial wall, including the intima, which is comprised of endothelial cells. Other preferred vectors are disclosed in Flugelman, et al., Circulation, v. 85, p. 1 1 10-1 117 (1992). Alternatively, naked FLIP nucleic acids or plasmids containing FLIP nucleic acids can be used in place of viral vectors to genetically modify the target cells to express functional FLIP polypeptides.
  • chemical/physical vectors may be used to deliver a FLIP molecule to a target cell and facilitate uptake thereby.
  • a "chemical/physical - 30 - vector” refers to a natural or synthetic molecule, other than those derived from bacteriological or viral sources, capable of delivering the isolated FLIP molecule to a cell.
  • a preferred chemical/physical vector of the invention is a colloidal dispersion system.
  • Colloidal dispersion systems include lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • a preferred colloidal system of the invention is a liposome.
  • Liposomes are artificial membrane vessels which are useful as a delivery vector in vivo or in vitro. It has been shown that large unilamellar vessels (LUV), which range in size from 0.2 - 4.0 ⁇ can encapsulate large macromolecules. RNA, DNA, and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem.
  • a liposome In order for a liposome to be an efficient gene transfer vector, one or more of the following characteristics should be present: (1) encapsulation of the gene of interest at high efficiency with retention of biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information.
  • Liposomes may be targeted to a particular tissue, such as the vascular cell wall, by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein.
  • Ligands which may be useful for targeting a liposome to the vascular wall include, but arc not limited to the viral coat protein of the Hcmagglutinating virus of Japan.
  • the vector may be coupled to a nuclear targeting peptide, which will direct the FLIP nucleic acid to the nucleus of the host cell.
  • Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTINTM and LIPOFECTACETM, which are formed of cationic lipids such as N-[l-(2, 3 dioleyloxy)- propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB).
  • LIPOFECTINTM and LIPOFECTACETM are formed of cationic lipids such as N-[l-(2, 3 dioleyloxy)- propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB).
  • DOTMA N-[l-(2, 3 dioleyloxy)- propyl]-N, N, N-trimethylammonium chloride
  • DDAB dimethyl dioctadecylammonium bromide
  • the preferred vehicle is a biocompatible micro particle or implant that is suitable for implantation into the mammalian recipient.
  • exemplary bioerodible implants that are useful in accordance with this method are described in PCT International application no. PCT/US/03307 (Publication No. WO 95/24929, entitled “Polymeric Gene Delivery System", claiming priority to U.S. patent application serial no. 213,668, filed March 15, - 31 -
  • PCT/US/0307 describes a biocompatible, preferably biodegradable polymeric matrix for containing an exogenous gene under the control of an appropriate promotor.
  • the polymeric matrix is used to achieve sustained release of the exogenous gene in the patient.
  • the FLIP nucleic acids described herein are encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in PCT/US/03307.
  • the polymeric matrix preferably is in the form of a micro particle such as a micro sphere (wherein the FLIP nucleic acid is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the FLIP nucleic acid is stored in the core of a polymeric shell).
  • polymeric matrix for containing the FLIP nucleic acid examples include films, coatings, gels, implants, and stents.
  • the size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix device is implanted.
  • the size of the polymeric matrix devise further is selected according to the method of deliver.' which is to be used, typically injection into a tissue or administration of a suspension by aerosol into the nasal and/or pulmonary areas.
  • the polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the devise is administered to a vascular surface.
  • the matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.
  • Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the FLIP nucleic acids of the invention to the subject.
  • Biodegradable matrices are preferred.
  • Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred.
  • the polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable.
  • the polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers.
  • the FLIP nucleic acids of the invention are delivered using the bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix.
  • exemplary synthetic polymers which can be used to form the biodegradable deliver ⁇ ' system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers - 32 - thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl
  • non-biodegradable polymers examples include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.
  • biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaecharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
  • Bioadhesive polymers of particular interest include bioerodible hydrogels described by H.S. Sawhney, C.P. Pathak and J.A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylatcs), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
  • the invention provides a composition of the above-described FLIP molecules for use as a medicament, methods for preparing the medicament and methods for the sustained release of the medicament in vivo.
  • the FLIP nucleic acid has the nucleic acid - 33 - sequence of SEQ. ID NOs. 1 or 3 or a functionally-equivalent fragment of SEQ. ID Nos. l or 3, such as a nucleic acid encoding a "FRAGMENT 1 " FLIP polypeptide or a nucleic acid encoding a "FRAGMENT 2" FLIP polypeptide as described herein.
  • the FLIP nucleic acid is operably linked to a gene expression sequence to permit expression of the FLIP polypeptide in the target cell.
  • the preferred FLIP polypeptide has the amino acid sequence of SEQ. ID NOs.
  • Compaction agents also can be used alone, or in combination with, a biological or chemical/physical vector of the invention.
  • a "compaction agent”, as used herein, refers to an agent, such as a histone, that neutralizes the negative charges on the nucleic acid and thereby permits compaction of the nucleic acid into a fine granule. Compaction of the nucleic acid facilitates the uptake of the nucleic acid by the target cell.
  • the compaction agents can be used alone, i.e., to deliver the isolated FLIP nucleic acid in a form that is more efficiently taken up by the cell or, more preferably, in combination with one or more of the above-described vectors.
  • the FLIP nucleic acids code for a FLIP polypeptide.
  • a FLIP polypeptide refers to a polypeptide that is coded for by a FLIP nucleic acid and that binds to a "death effector domain” (DED) but does not activate downstream proteases.
  • the FLIP polypeptides inhibit Fas ligand-mediated apoptosis.
  • the FLIP polypeptide inhibits Fas ligand-mediated apoptosis in an endothelial cell and, more particularly, inhibits apoptosis in a vascular endothelial cell.
  • FLIP polypeptides are useful for inhibiting Fas ligand-mediated vascular endothelial cell apoptosis in vivo and in vitro.
  • the preferred FLIP polypeptides of the invention have the amino acid sequence of SEQ. ID NOs. 2 or 4 or are functionally equivalent fragments of SEQ. ID Nos.2 or 4.
  • FLIP polypeptides further embrace functionally equivalent variants, and analogs of SEQ. ID NO. 2 or 4, provided that the fragments, variants, and analogs are capable of binding to a death effector domain and, optionally, inhibiting Fas ligand-mediated apoptosis.
  • the invention also embraces proteins and peptides coded for by any of the foregoing FLIP nucleic acids.
  • a "functionally equivalent variant" of SEQ. ID NO. 2 or 4 binds to a death domain and, optionally, inhibits Fas ligand-induced apoptosis.
  • a functionally equivalent variant of SEQ. ID NO. 2 or 4 is capable of inhibiting such apoptosis in an endothelial cell (e.g., a vascular endothelial cell) in vitro or in vivo.
  • An in vitro apoptosis assay (see, e.g., the apoptosis assay provided in the Examples) can be used as a screening assay to measure the ability of a polypeptide to inhibit Fas ligand-mediated apoptosis in a vascular endothelial cell in vitro and is predictive of the ability of the polypeptide to inhibit apoptosis of vascular endothelial cells in vivo.
  • binding assays can be used to select such functionally equivalent variants, e.g., by selecting variant that bind to a death domain in an in vitro assay.
  • binding assays to select protein-type binding cognates are known to those of ordinary skill in the art.
  • Exemplary "functionally equivalent variants" of SEQ. ID. Nos. 2 or 4 includes fragments of SEQ. ID. Nos. 2 or 4, as well as polypeptide analogs of SEQ. ID. Nos. 2 or 4 which contain conservative amino acid substitutions, provided that the polypeptide variants and analogs are capable of binding to a death effector domain and, preferably, inhibiting Fas ligand-mediated apoptosis of vascular endothelial cells.
  • the preferred FLIP nucleic acids of the invention encode the FLIP having the amino acid sequence of SEQ. ID NO. 2, the complete coding sequence of the gene encoding the human FLIP.
  • This "intact" human FLIP polypeptide contains two death effector domains and four caspase-like domains as described above.
  • the invention also embraces compositions containing and methods for using "functionally equivalent fragments" of the FLIP polypeptide, namely, "FRAGMENT 1 polypeptides” and “FRAGMENT 2 polypeptides", and so forth as described herein.
  • polypeptides are fragments of SEQ. ID Nos.2 or 4. No prior use for the FRAGMENT 1 polypeptides and
  • FRAGMENT 2 polypeptides disclosed here has been proposed. Accordingly, one particular aspect of the invention relates to such FRAGMENT 1 polypeptides, nucleic acids encoding same, complements of said nucleic acids, vectors containing said nucleic acids, host cells containing said vectors, and methods for using the foregoing compositions.
  • Alternative embodiments include FLIP polypeptides that are identical in amino acid sequence to SEQ. ID Nos.2 or 4 and fragments of SEQ. ID Nos.2 or 4, but which differ from SEQ. ID Nos.2 or 4 in having one or more amino acid substitutions in the caspase-like domain proteolytic cleavage site, i.e., amino - 35 - acid Asp 376 in SEQ. ID NO. 1. See also, Irmler et al., Nature supra., which describes the cleavage of FLIP-L at Asp 376 to yield a molecule that reportedly binds with greater affinity to FLICE.
  • amino acids include substitutions made amongst amino acids with the following groups: (1) M,I,LN; (2) F,Y,W; (3) K,R,H; (4) A,G; (5) S,T; (6) Q, ⁇ ; and, (7) E,D.
  • Fusion proteins in which a peptide of the invention is coupled to a solid support (such as a polymeric bead), a carrier molecule (such as keyhole limpet hemocyanin), or a reporter group (such as radiolabel or other tag), also are embraced within the invention.
  • the isolated FLIP molecules of the invention are administered in therapeutically effective amounts.
  • a therapeutically effective amount means that amount necessary to delay the onset of, inhibit the progression of, or halt altogether the particular condition being treated.
  • a therapeutically effective amount will vary with the subject's age, condition, and sex, as well as the nature and extent of the disease in the subject, all of which can be determined by one of ordinary skill in the art.
  • the dosage may be adjusted by the individual physician or veterinarian, particularly in the event of any complication.
  • a therapeutically effective -amount typically varies from 0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg, and most preferably from about 0.2 mg//kg to about 20 mg/kg, in one or more dose administrations daily, for one or more days.
  • the therapeutically effective amount of the isolated FLIP molecule is that amount effective to inhibit vascular wall inflammation as determined by, for example, standard tests known in the art. It is believed that the FLIP molecules inhibit apoptosis in the target cells by interfering with the Fas ligand-Fas receptor signaling pathway. For example, TUNEL staining, and the appearance of condensed chromatin and other morphological features characteristic of apoptosis in electron micrographs can be used to assess apoptosis in vascular endothelial and other cell types. -, . PCT/US99/03558
  • the isolated FLIP molecule is administered to the subject in combination with a method for treating an arteriosclerotic condition.
  • An arteriosclerotic condition is a term of art that refers to classical atherosclerosis, transplant arteriosclerosis, accelerated atherosclerosis, atherosclerotic lesions and other physiological conditions characterized by undesirable vascular wall inflammation. See, e.g., Harrisons, Principles of Internal Medicine (McGraw Hill, Inc., New York) for a more detailed description of these conditions.
  • the method for treating an arteriosclerotic condition may be a surgical method, an agent for treating restenosis, a method involving a drug therapy (e.g., gene therapy) or a combination of the foregoing.
  • Surgical methods for treating an arteriosclerotic condition include procedures such as bypass surgery, atherectomy, laser procedures, ultrasonic procedures, and balloon angioplasty.
  • the isolated FLIP molecule is administered to a subject in combination with a balloon angioplasty procedure.
  • a balloon angioplasty procedure involves inserting a catheter having a deflated balloon into an artery. The deflated balloon is positioned in proximity to the atherosclerotic plaque and is inflated such that the plaque is compressed against the arterial wall. As a result, the layer of endothelial cells on the surface of the artery is disrupted.
  • the isolated FLIP molecule is attached to the balloon angioplasty catheter in a manner which permits release of the isolated FLIP molecule to the remaining endothelium at the site of the atherosclerotic plaque.
  • the isolated FLIP molecule may be attached to the balloon angioplasty catheter in accordance with standard procedures known in the art.
  • the isolated FLIP molecule may be stored in a compartment of the balloon angioplasty catheter until the balloon is inflated, at which point it is released into the local environment.
  • the isolated FLIP molecule may be impregnated on the balloon surface, such that it contacts the cells of the arterial wall as the balloon is inflated.
  • the FLIP molecule also may be delivered in a perforated balloon catheter such as those disclosed in Flugelman, et al., Circulation, v. 85, p. 1 110-1117 (1992). See, also, e.g., published PCT Patent Application WO 95/23161, for an exemplary procedure for attaching a therapeutic protein to a balloon angioplasty catheter. This procedure can be modified using no more that routine experimentation to attach a therapeutic nucleic acid or polypeptide to the balloon angioplasty catheter. Additionally, the FLIP molecule may be administered with an agent for treating or preventing clinically significant restenosis, which often occurs following balloon angioplasty procedures. Restenosis is narrowing of the artery which occurs in 25% to 50% of patients within - 37 -
  • FLIP molecules of the invention is believed to be useful for treating vascular remodeling, in general, and in-stent restenosis, in particular, through its ability to promote endothelial cell viability and preserve the integrity of the endothelium.
  • a preferred agent for preventing restenosis, in combination with the FLIP molecule is a stent.
  • Stents are discussed in a review article by Topol, E. J., the contents of which are hereby incorporated by reference (Topol, E. J., N. E. J. Med. 331: 539-41 (1994)).
  • Stents include, for example, the Gianturco-Roubin stent and the Palmaz-Schatz stent.
  • the arteriosclerotic conditions also can be treated by a nonsurgical method such as a drug therapy.
  • drugs have been used to treat various aspects of an arteriosclerotic condition.
  • drugs have been used to treat physiological events, such as hypertension and excessive cholesterol accumulation, which are believed to contribute to the formation of atherosclerotic plaques.
  • Other drugs influence the site of injury by breaking up or reducing the size of atherosclerotic plaques, and/or increasing the strength of the arterial wall.
  • the isolated FLIP molecule may be administered in conjunction with either or a combination of drugs which inhibit the physiological events contributing to arteriosclerosis or drugs which function directly to reduce the local site of injury associated with atherosclerosis.
  • Drug therapies which have been found to be useful in treating the physiological events contributing to the development of the atherosclerotic injury, include, but are not limited to, the following drugs: diuretics, antiadrenergic agents, vasodilators, calcium channel antagonists, HMG-CoA reductase inhibitors, angiotensin-converting enzyme (ACE) inhibitors, angiotensin
  • Diuretics include thiazidcs, e.g., h'ydrochlorothiazide; loop acting diuretics, e.g., furosemide; potassium-sparing, e.g., spironolactone, triamterene, and amiloride.
  • Antiadrenergic agents include clonidine; guanabenz; guanfacinc; mcthyldopa; trimcthapajn; Rauwolfia alkaloids, e.g., reserpine; guanethidine; guanadrel; phentolamine; phcnoxybcnzamine; prazosin; terazosin; propranolol; metoprolol; nadolol; atenolol; timolol; timdolol; acebutol ⁇ l; and labetalol. - 38 -
  • Vazodilators include hydralazine; minoxidil; diazoxide; and nitroprusside.
  • Calcium channel antagonists include nisadipine; diltiazcn; and vcrapamil.
  • Angiotensin II antagonists are compounds which interfere with the activity of angiotensin II by binding to angiotensin II receptors and interfering with its activity.
  • Angiotensin II antagonists are well known and include peptide compounds and non-peptide compounds. Most angiotensin II antagonists are slightly modified congeners in which agonist activity is attenuated by replacement of phenylalanine in position 8 with some other amino acid; stability can be enhanced by other replacements that slow degeneration in vivo.
  • angiotensin II antagonists include: peptidic compounds (e.g., saralasin, [(San')(Val 5 )(Ala 8 )] angiotensin -(1-8) octapeptide and related analogs); N-substituted imidazole-2-one (US Patent Number 5,087,634); imidazole acetate derivatives including 2-N-butyl-4-chloro-l -(2-chlorobenzile) imidazole-5-acetic acid (see Long et al., J. Pharmacol. Exp. Ther.
  • peptidic compounds e.g., saralasin, [(San')(Val 5 )(Ala 8 )] angiotensin -(1-8) octapeptide and related analogs
  • N-substituted imidazole-2-one US Patent Number 5,087,634
  • imidazole acetate derivatives including 2-N-buty
  • ACE is an enzyme which catalyzes the conversion of angiotensin I to angiotensin II.
  • ACE inhibitors include amino acids and derivatives thereof, peptides, including di and tri peptides and antibodies to ACE which intervene in the renin-angiotensin system by inhibiting the activity of ACE, thereby reducing or eliminating the formation of prcssor substance angiotensin II.
  • ACE inhibitors have been used medically to treat hypertension, congestive heart failure, myocardial infarction and renal disease.
  • Classes of compounds known to be useful as ACE inhibitors include - 39 - acylmercapto and mercaptoalkanoyl prolines such as captopril (US Patent Number 4,105,776) and zofenopril (US Patent Number 4,316,906), carboxyalkyl dipeptides such as enalapril (US Patent Number 4,374,829), lisinopril (US Patent Number 4,374,829), quinapril (US Patent Number 4,344,949), ramipril (US Patent Number 4,587,258), and perindopril (US Patent Number s 4,508,729), carboxyalkyl dipeptide mimics such as cilazapril (US Patent Number 4,512,924) and benazapril (US Patent Number 4,410,520), phosphinylalkanoyl prolines such as fosinopril (US Patent Number 4,337,201) and trandolopril.
  • captopril US Patent Number
  • Renin inhibitors are compounds which interfere with the activity of renin. Renin inhibitors include amino acids and derivatives thereof, peptides and derivatives thereof, and antibodies to o renin. Examples of renin inhibitors that are the subject of United States patents are as follows: urea derivatives of peptides (US Patent Number 5,116,835); amino acids connected by nonpeptide bonds (US Patent Number 5,114,937); di and tri peptide derivatives (US Patent Number 5,106,835); amino acids and derivatives thereof (US Patent Numbers 5,104,869 and 5,095,1 19); diol sulfonamides -and sulfinyls (US Patent Number 5,098,924); modified peptides (US Patent 5 Number 5,095,006); peptidyl beta-am inoacyl aminodiol carbamates (US Patent Number
  • the isolated FLIP molecule may be administered to a subject in combination with an antisense oligonucleotide that is targeted to vascular smooth muscle cells and that selectively hybridizes to cell cycle regulatory molecules, such as c-myb, cdc2, cdk2, PCNA, and c-myc under physiological conditions.
  • cell cycle regulatory molecules such as c-myb, cdc2, cdk2, PCNA, and c-myc under physiological conditions.
  • Such antisense oligonucleotides o can function as cytostatic or cytotoxic agent, depending upon the relative amounts of the antisense oligonucleotides that are delivered to the vascular smooth muscle cell and the importance of the particularly targeted cell cycle regulatory molecule to cell growth, proliferation and survival.
  • compositions also arc capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • Pharmaceutically acceptable further means a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism.
  • the characteristics of the carrier will depend on the route of administration.
  • Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers; stabilizers, solubilizers, and other materials which are well known in the art.
  • Such modes of administration include oral, rectal, topical, nasal, interdermal, or parenteral routes.
  • parenteral includes subcutaneous, intravenous, intramuscular, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. They could, however, be preferred in emergency situations. Oral administration will be preferred for prophylactic treatment because of the convenience to the patient as well as the dosing schedule.
  • compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a prcdctemiincd amount of the FLIP molecule.
  • Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the FLIP molecules - 43 - described above, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include the above-described polymeric systems, as well as polymer base systems such as poly(lactide- glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109.
  • Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • Specific examples include, but are not limited to: (a) erosional systems in which an agent of the invention is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,854,480, 5,133,974 and 5,407,686.
  • pump-based hardware delivery systems can be used, some of which are adapted for implantation.
  • the isolated FLIP molecule may be administered alone or in combination with the above- described drug therapies by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intra-cavity, subcutaneous, or transdermal.
  • direct administration to the vessel injury site such as by administration in conjunction with a balloon angioplasty catheter, is preferred.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the FLIP nucleic acids can be administered to the subject (any mammalian recipient) using the same modes of administration that currently are used for gene therapy in humans (e.g., adenovirus-mediated gene therapy).
  • the FLIP nucleic acid (contained in, or associated with, an appropriate vector) is administered to the mammalian recipient by balloon angioplasty catheter (described above) or intra- vascular injection.
  • balloon angioplasty catheter described above
  • intra- vascular injection a transfection and transduction tecliniques as well as appropriate expression vectors are well known to those of ordinary skill in the art, some of which are described in PCT application WO95/00654.
  • a vector containing a FLIP nucleic acid is delivered to a site of vascular injury in a subject who is a candidate for such gene therapy. Then, the vector genetically modifies the vascular endothelial cells in vivo with DNA (RNA) encoding a FLIP polypeptide of the invention.
  • RNA DNA
  • Such genetically modified vascular endothelial cells are expected to inhibit Fas ligand-mediated apoptosis of vascular endothelial cells in vivo.
  • Oxidized LDL for use in the screening assay is prepared by oxidizing LDL either chemically, for example, by incubating LDL at 37°C for 24 hours, at about 0.2 mg protein/ml in PBS with about 20 ⁇ M CuS0 4 or via a cell mediated process, e.g., by incubating LDL in the presence of endothelial cells al 37° C for 24 hours, at a concentration of about 0.07 - 0.1 mg protein/ml, in serum free medium containing 12 ⁇ M CuS0 4 .
  • An increase in the number of cells - 45 - that survive indicates that the putative therapeutic agent inhibits oxidized lipid-mediated down regulation (direct or indirect) of FLIP expression in the cell.
  • the assay optionally includes one or more negative controls, e.g., cells of the same cell type which have not received a FLIP molecule but which have been exposed to Fas ligand.
  • the method also involves the step of contacting the FLIP molecule with a detection reagent that selectively binds to the FLIP molecule to detect or measure the amount of the FLIP molecule in the "test" cell.
  • the FLIP molecule may optionally be isolated from the vascular endothelial or other cell prior to contacting the isolated FLIP molecule with the detection reagent.
  • the detection reagent can be a nucleic acid that selectively hybridizes to the FLIP mRNA or cDNA.
  • the "test" cell is contacted with the detection reagent under conditions that permit selective hybridization of the nucleic acid to the FLIP mRNA or cDNA.
  • the preferred nucleic acid for this embodiment is a nucleic acid sequence having SEQ. ID. No. 1 or a functionally equivalent fragment thereof.
  • the FLIP molecule that is being assayed can be a FLIP polypeptide and the detection reagent can be an antibody that selectively binds to the FLIP protein.
  • the FLIP polypeptide can be contacted with the detection reagent under conditions that permit selective binding of a FLIP antibody to the FLIP polypeptide.
  • the FLIP nucleic acid of the invention can be used to prepare a non-human transgenic animal that can be used, for example, as an animal model (e.g., a FLIP knockout animal) of excessive smooth muscle cell proliferation.
  • a "transgenic animal” is an animal having cells that contain DNA which has been artificially inserted into a cell, which DNA becomes part of the genome of the animal which develops from that cell.
  • Preferred transgenic animals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogs and cats. Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River (Wilmington, MA), Taconic (Germantown, NY), Harlan Sprague Dawley (Indianapolis, IN), etc.
  • Transgenic animals having a particular property associated with a particular disease can be used to study the affects of a variety of drugs and treatment methods on the disease, and thus serve as genetic models for the study of a number of human diseases.
  • the invention contemplates the use of FLIP knockout and transgenic animals as models for the study of disorders of vascular blood vessels, such as arteriosclerosis as well as for the study of transplant arteriosclerosis.
  • DNA can be injected into the pronucleus of a fertilized egg before fusion of the male and female pronuclei, or injected into the nucleus of an embryonic cell (e.g., the nucleus of a two-cell embryo) following the initiation of cell division.
  • an embryonic cell e.g., the nucleus of a two-cell embryo
  • transgenic animals An alternative method for producing transgenic animals involves the incorporation of the desired gene sequence into a virus which is capable of affecting the cells of a host animal. See e.g., Elbrecht et al., Molec. Cell. Biol. 7: 1276 (1987); Lacey et al, Nature 322: 609 (1986);
  • Embryos can be infected with viruses, especially retroviruses, modified to carry the nucleotide sequences of the invention which encode FLIP proteins or sequences which disrupt the native FLIP gene to produce a knockout animal.
  • Another method for producing transgenic animals involves the injection of pluripotent embryonic stem cells into a blastocyst of a developing embryo.
  • Pluripotent stem cells derived from the inner cell mass of the embryo and stabilized in culture can be manipulated in culture to incorporate nucleotide sequences of the invention.
  • a transgenic animal can be produced from such cells through implantation into a blastocyst that is implanted into a foster mother and allowed to come to term. See e.g., Robertson et al., Cold Spring Harbor Conference Cell Proliferation 10: 647 (1983); Bradley et al., Nature 309: 255 (1984); Wagner et al., Cold Spring
  • mice are induced to superovulate.
  • Females are placed with males, and the mated females are sacrificed by C0 2 asphyxiation or cervical dislocation and embryos are recovered from excised oviducts. Surrounding cumulus cells are removed. Pronuclear embryos arc then washed and stored until the time of injection. Randomly cycling adult female mice are paired with vasectomized males. Recipient females are mated at the same time as donor females. Embryos then are transferred - 47 - surgically. The procedure for generating transgenic rats is similar to that of mice. See Hammer et al., Ce/ , 63:1099-1112 (1990).
  • a clone containing the sequence(s) of the invention is co-transfected with a gene encoding resistance.
  • the gene encoding neomycin resistance is physically linked to the sequence(s) of the invention.
  • DNA molecules introduced into ES cells can also be integrated into the chromosome through the process of homologous recombination.
  • Capecchi Science, 244: 1288-1292 (1989).
  • Methods for positive selection of the recombination event (e.g.. neo resistance) and dual positive-negative selection (e.g., neo resistance and gangcyclovir resistance) and the subsequent identification of the desired clones by PCR have been described by Capecchi, supra and Joyner et al., Nature, 338: 153-156 (1989).
  • the final phase of the procedure is to inject targeted ES cells into blastocysts and to transfer the blastocysts into pseudopregnant females.
  • the resulting chimeric animals are bred and the offspring are analyzed by Southern blotting to identify individuals that carry the transgene.
  • Inactivation or replacement of the endogenous FLIP gene can be achieved by a homologous recombination system using embryonic stem cells.
  • the resultant transgenic non-human mammals having a knockout FLIP characteristic may ' be used as a model for atherosclerosis.
  • Vascular endothelial cells which are exposed to oxidized lipid may be predisposed to apoptosis and thus, result in vascular wall inflammation and produce an atherosclerotic phenotype.
  • a normal or mutant version of FLIP can be inserted into the mouse germ line to produce transgenic animals which constitutively or inducible express the normal or mutant form of FLIP.
  • transgenic animals which constitutively or inducible express the normal or mutant form of FLIP.
  • FasL vascular endothelial cells express FasL, which can function to induce apoptotic cell death in adherent leukocytes.
  • FasL FasL
  • adherent leukocytes adherent leukocytes
  • endothelial cells are resistant to Fas-mediated apoptosis although they express detectable Fas on their cell surface (Richardson et al., 1994; Sata and Walsh, 1998 ).
  • HUVECs treated with OxLDL displayed characteristics of apoptosis including cell shrinkage and nuclear condensation, DNA fragmentation, and decreased mitochondrial function (Fig. 2).
  • Apoptosis was markedly attenuated when cultures were co-incubated with a neutralizing anti-FasL antibody, demonstrating that FasL is essential for the induction of endothelial cell apoptosis by OxLDL.
  • Similar observations were also made with HAECs (not shown).
  • OxLDL-treated endothelial cells protected from death attained a distinctive elongated cell morphology, indicating that other actions of OxLDL are not blocked by the neutralizing anti-FasL antibody.
  • FasL and Fas can become dramatically sensitized to the Fas-mediated apoptosis in response to specific stimuli. It is well established that T lymphocyte number is controlled by a delayed sensitization to Fas-mediated apoptosis following activation (Klas, 1993). Perturbations in the Fas/FasL cell suicide pathway may also be important in determining the viability of transformed cells (Hueber et al., 1997; Muller et al., 1997), and there is increasing evidence that cancer cell sensitivity to Fas-mediated apoptosis is a key feature of tumor progression (Hahne et al., 1996; O'Connell et al., 1996; Strand et al., 1996).
  • Fas-mcdiatcd cell suicide may also have a role in determining the viability of endothelial cells to injurious agents.
  • injuries to endothelium trigger inflammatory-fibroproliferative processes in the vessel wall (Ross, 1993)
  • OxLDL OxLDL to sensitize ECs to FasL/Fas-mediated suicide may contribute to the accelerated - 50 - atherosclerosis seen in patients with hyperlipemia.
  • Our data also suggest that alterations in Fas- mediated cell suicide may have a more widespread role in disease processes than was previously appreciated.
  • HUVECs were detached from the culture plate with 0.5% EDTA and incubated with an anti-FasL polyclonal antibody (C-20, Santa Cruz) (filled curve) or with a rabbit lgG (open curve) in PBS with 10% FBS, followed by incubation with an FITC-conjugated anti-rabbit Ig antibody. Immunofluorescence staining was analyzed by FACS (fluorescence activated cell sorter) (Becton
  • OxLDL OxLDL-induced apoptosis and morphological changes
  • HAECs (-90%) confluent) were incubated with or without OxLDL (300 ⁇ g protein/ml) in the absence and presence of an anti-FasL antibody (10 ⁇ g/ml, 4H9, MBL, Nagoya) that is capable of neutralizing FasL (Tanaka et al., 1996).
  • an anti-FasL antibody 10 ⁇ g/ml, 4H9, MBL, Nagoya
  • FasL FasL
  • Endothelial cells express Fas receptor but are not normally susceptible to Fas ligand-mediated cell death; however, dysfunctional endothelial cells are susceptible to Fas ligand- mediated apoptosis.
  • an Adeno-FLIP construct (Fig. 7) is prepared as described below and utilized to characterize the differential effects of FLIP expression on endothelial cell viability in vitro (in the presence or absence of Fas ligand, with or without oxidized lipid).
  • This construct also is used to assess the effects of increased apoptosis on lesion formation in the rat carotid and rabbit iliac models of vascular injury. It is believed that the endothelial cells that are engineered to express FLIP behave as normal endothelial cells even in the presence of oxidized lipid and, in this manner, dysfunctional endothelial cells (susceptible to Fas ligand-mediated apoptosis) can be converted into normal, Fas ligand-resistant cells.
  • the resulting plasmids are linearized by restriction enzyme digestion and cotransfected in 293 cells with large Clal fragment of the Ad5 dl324 viral DNA (Stratford-Perricaudet, L.D., et al., 1993, J. Clin. Invest. 90:626-630).
  • the resulting replication-defective recombinant adenoviral constructs are purified from isolated plaques.
  • the viral preparations are purified by two CsCl gradient centrifugations, dialyzed against buffer containing 10 mM Tris-Cl pH 7.5, 1 mM MgCl 2 and 135 mM NaCl and stored at -80°C in 10% glycerol.
  • Viral titer is determined by plaque assay on 293 cells (Graham, F.L., and A.J. van der Eb, 1973, Virology 52:456-463) and expressed as plaque forming units (pfu) per ml.
  • an inducihly expressed Adeno-FLIP construct is made by subcloning a FLIP cDNA (based upon human FLIP-L or FLIP-S sequences as shown in SEQ. ID Nos. 1 or 3), downstream from the tctracycline-responsive element (TRE) of the Clontech, (Palo Alto, - 53 -

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Abstract

La présente invention concerne une méthode de traitement des états associés à l'inflammation des parois vasculaires, en particulier l'artériosclérose et les troubles vasculaires. Cette méthode consiste à administrer à des sujets ayant besoin d'un tel traitement une dose efficace d'une molécule FLIP.
PCT/US1999/003558 1998-02-20 1999-02-19 Compositions a base de flip cellulaire utilisees dans le traitement des troubles arteriosclereux WO1999042570A1 (fr)

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AT413485B (de) * 2001-04-09 2006-03-15 Hemoteq Gmbh Mit einem pharmazeutikum beschichteter stent

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