EP1601696A1 - Compositions et procedes permettant d'inhiber l'inflammation des parois vasculaires et la formation d'hyperplasie de neo-intima - Google Patents

Compositions et procedes permettant d'inhiber l'inflammation des parois vasculaires et la formation d'hyperplasie de neo-intima

Info

Publication number
EP1601696A1
EP1601696A1 EP04717875A EP04717875A EP1601696A1 EP 1601696 A1 EP1601696 A1 EP 1601696A1 EP 04717875 A EP04717875 A EP 04717875A EP 04717875 A EP04717875 A EP 04717875A EP 1601696 A1 EP1601696 A1 EP 1601696A1
Authority
EP
European Patent Office
Prior art keywords
composition
patient
nucleic acid
vector
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04717875A
Other languages
German (de)
English (en)
Inventor
Kensuke 101 Aquacourt 2bankan EGASHIRA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Anges Inc
Original Assignee
Anges MG Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Anges MG Inc filed Critical Anges MG Inc
Publication of EP1601696A1 publication Critical patent/EP1601696A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to gene therapy, and more specifically to compositions and methods for inhibiting or treating formation of neointimal hyperplasia using a gene encoding a soluble fragment of fms-like tyrosine kinase-1 (Flt-1).
  • Neointimal hyperplasia is a major cause of restenosis after coronary intervention (Libby P, Ganz P. Restenosis revisited— new targets, new therapies. N Engl J Med. 1997;337:418-9; and Topol EJ, Serruys PW. Frontiers in interventional cardiology. Circulation. 1998;98:1802-20).
  • Vascular endothelial growth factor (VEGF) and its receptors are upregulated in vascular inflammatory and proliferative disorders such as atherosclerosis and restenosis (Shibata M, Suzuki H, ⁇ akatani M, Koba S, Geshi E, Katagiri T, Takeyama Y.
  • vascular endothelial growth factor and flt-1 in the process of neointimal proliferation in pig coronary arteries following stent implantation. Histochem Cell Biol.
  • VEGF Vascular endothelial growth factor
  • VEGF is thought to protect the artery from such disorders by inducing endothelial regeneration and improving endothelial function mainly through the endothelial type 2 receptor Flk-1, and VEGF gene transfer or administration of its protein induces endothelial regeneration and attenuates ⁇ IH after endothelial injury (Baumgartner I, Isner JM. Somatic gene therapy in the cardiovascular system. Annu Rev Physiol. 2001;63:427-50).
  • VEGF endothelial regeneration and attenuates ⁇ IH after endothelial injury
  • Isner JM Somatic gene therapy in the cardiovascular system.
  • VEGF vascular endothelial growth factor
  • ICM-1 intercellular adhesion molecule 1
  • VCAM-1 vascular cell adhesion molecule 1
  • E- selectin through nuclear factor-kappa B activation in endothelial cells. J Biol Chem. 2001;276:7614-20
  • MCP-1 monocyte chemoattractant
  • VEGF protein enhances atherogenesis by inducing monocyte infiltration and activation (Celletti FL, Waugh JM, Amabile PG, Brendolan A, Hilfiker PR, Dake MD.
  • Vascular endothelial growth factor enhances atherosclerotic plaque progression. Nat Med. 2001;7:425-9).
  • VEGF might promote migration of vascular smooth muscle cells though Flt-1 (Grosskreutz CL, Anand-Apte B, Duplaa C, Quinn TP, Terman Bl, Zetter B, D'Amore PA.
  • Flt-1 acts as an important mediator of chemotaxis through VCAM-1, ICAM-1, and MCP-1 (Barleon B et al., supra, Kim I et al., supra, and Marumo T et al., supra).
  • Luttun et al reported that treatment with anti-Fit- 1 antibody attenuated the development of experimental tumor angiogenesis, arthritis, and atherosclerosis (Luttun A, Tjwa M, Moons L, Wu Y, Angelillo-Scherrer A, Liao F, Nagy JA, Hooper A, Priller J, De Klerck B, Compernolle V, Daci E, Bohlen P, Dewerchin M, Herbert JM, Fava R, Matthys P, Carmeliet G, Collen D, Dvorak HF, Hicklin DJ, Carmeliet P.
  • VEGF vascular endothelial growth factor
  • WO98/13071 discloses gene therapy methodology for inhibition of primary tumor growth and metastasis by gene transfer of a nucleotide sequence encoding a soluble form of a VEGF tyrosine kinase receptor to a mammalian host.
  • An object of the present invention is to clarify the role of VEGF in the development of NIH and to provide compositions and methods for inhibiting inflammation of vessel walls and/or formation of NIH.
  • the present inventor previously demonstrated that intramuscular transfection of the sFlt-1 gene effectively and specifically blocks VEGF signaling, and thus quenches VEGF activity in vivo (Zhao Q, Egashira K, Inoue S, Usui M, Kitamoto S, Ni W, Ishibashi M, Hiasa Ki K, Ichiki T, Shibuya M, Takeshita A.
  • Vascular endothelial growth factor is necessary in the development of arteriosclerosis by recruiting/activating monocytes in a rat model of long-term inhibition of nitric oxide synthesis. Circulation. 2002;105:1110-5, and Goldman CK et al. supra). Subsequently, the present inventor investigated the role of VEGF in the pathogenesis of NIH following cuff-induced periarterial injury in hypercholesterolemic mice.
  • This cuff model was chosen because cuff placement in the presence of hypercholesterolemia offers the advantage of inducing reproducible site-controlled NIH and remodeling, and also the cuff-induced injury triggers vascular inflammation and induces neointimal lesions that are similar to the restenotic and atherosclerotic lesions observed in humans (Lardenoye JH, Delsing DJ, de Vries MR, Deckers MM, Princen HM, Havekes LM, van Hinsbergh VW, van Bockel JH, Quax PH. Accelerated atherosclerosis by placement of a perivascular cuff and a cholesterol-rich diet in ApoE*3Leiden transgenic mice. Circ Res.
  • Perivascular inflammation has a major role in the pathogenesis of cuff-induced NIH (Egashira K, Zhao Q, Kataoka C, Ohtani K, Usui M, Charo IF, Nishida K, Inoue S, Katoh M, Ichiki T, Takeshita A. Importance of monocyte chemoattractant protein-1 pathway in neointimal hyperplasia after periarterial injury in mice and monkeys. Circ Res. 2002;90:1167-72, and Wu L, Iwai M, Nakagami H, Li Z, Chen R, Suzuki J, Akishita M, de Gasparo M, Horiuchi M.
  • VEGF is conventionally thought to be an endothelial cell-specific growth factor and to attenuate vascular disease by inducing endothelial proliferation and regeneration mainly through the endothelial type 2 receptor Flk-1 (Baumgartner I et al., supra). Recent evidence, however, suggests that functional VEGF receptors are expressed in injured arterial walls in cells other than endothelial cells. Therefore, the relative effects of Flt-1- versus Flk-1-mediated action are likely to depend on the relative expression of Flt-1 and Flk-1 in target cells. The present inventor herein demonstrate that Flt-1 was increased in lesional monocytes and medial smooth muscle cells at early stages and in neointimal and medial smooth muscle cells at later stages.
  • neointimal and medial cells were bone marrow-derived progenitor cells that differentiated into smooth muscle cells and endothelial cells in vascular lesions of models of post-angioplasty restenosis, transplant-associated arteriosclerosis, and hyperlipidemia-induced atherosclerosis (Sata M, Saiura A, Kunisato A, Tojo A, Okada S, Tokuhisa T, Hirai H, Makuuchi M, Hirata Y, Nagai R. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med. 2002;8:403-9).
  • Flt-1 is an important mediator of stem cell recruitment and mobilization (Luttun A, Tjwa M, Moons L, Wu Y, Angelillo-Scherrer A, Liao F, Nagy JA, Hooper A, Priller J, De Klerck B, Compernolle V, Daci E, Bohlen P, Dewerchin M, Herbert JM, Fava R, Matthys P, Carmeliet G, Collen D, Dvorak HF, Hicklin DJ, Carmeliet P. Revascularization of ischemic tissues by P1GF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Fit 1. Nat Med. 2002;8:831-40).
  • the present inventor herein demonstrate that cells in the neointima or media rarely expressed a marker of bone marrow origin in bone marrow-transplanted mice after cuff placement, indicating a minor contribution of bone marrow-derived cells to neointimal formation in this model.
  • VEGF-mediated inflammation To gain insight into the mechanism of VEGF-mediated inflammation after cuff placement, the present inventor assessed gene expression of various inflammatory genes. sFlt-1 gene transfer attenuated increased gene expression of inflammatory cytokines, adhesion molecules, chemokines, and chemokine receptors (Figure 5). These data are consistent with prior reports demonstrating that VEGF induces adhesion molecules (VCAM-1 and ICAM-1) or MCP-1 in endothelial cells in vitro (Kim I et al., supra and Marumo T et al., supra).
  • sFlt-1 gene transfer attenuated increased VEGF and Flt-1 gene expression, indicating that VEGF regulates its activity by an autocrine loop mechanism within diseased arterial wall cells such as smooth muscle cells, endothelial cells, and lesional monocytes.
  • a positive feedback effect of VEGF is supported by prior studies that demonstrated enhanced VEGF production by monocytes through Flt-1 stimulation (Bottomley MJ, Webb ⁇ J, Watson CJ, Holt L, Bukhari M, Denton J, Freemont AJ, Brenchley PE.
  • Placenta growth factor (P1GF) induces vascular endothelial growth factor (VEGF) secretion from mononuclear cells and is co-expressed with VEGF in synovial fluid.
  • P1GF Placenta growth factor
  • VEGF vascular endothelial growth factor
  • VEGF and its receptor signals appear to be essential for the development of early inflammation as well as late NIH after cuff- induced perivascular cuff injury.
  • VEGF is likely to promote NIH by activating and recruiting monocytes and vascular smooth muscle cells.
  • the data shown herein support the notion that VEGF works as a pro-inflammatory and pro-arteriosclerotic factor after cuff- induced periarterial injury.
  • the present invention provides:
  • composition comprising a nucleic acid encoding soluble Flt-1 (sFlt-1) and a pharmaceutically acceptable carrier, wherein said nucleic acid expresses sFlt-1 in an amount effective to inhibit or treat inflammation of vessel wall and/or formation of neointimal hyperplasia;
  • composition of (1) wherein the nucleic acid is inserted in a vector
  • composition of (2) wherein the vector is selected from the group consisting of a plasmid, an adenovirus vector, and a Hemagglutinating virus of Japan envelope (HVJ-E) vector; (4) the composition of (2), wherein the vector is a eukaryotic expression plasmid;
  • (21) a method for inhibiting or treating inflammation of vessel wall and/or formation of neointimal hyperplasia, comprising administration of a nucleic acid encoding soluble Flt-1 (sFlt-1) to a patient in need thereof; (22) the method of (21), wherein the nucleic acid is inserted in a vector;
  • the vector is selected from the group consisting of a plasmid, an adenovirus vector, and a Hemagglutinating virus of Japan envelope (HVJ-E) vector;
  • nucleic acid encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 2 which has one or more amino acid substitution, deletion, addition, and/or insertion, wherein said polypeptide is functionally equivalent to and has at least 65% identity to a polypeptide comprising amino acid sequence of SEQ ID NO: 2;
  • a soluble Flt-1 (sFlt-1) gene means a polynucleotide or nucleic acid that encodes and expresses an sFlt-1 protein.
  • a polynucleotide or nucleic acid may be DNA or RNA. It can be obtained by isolation from a natural source or by synthesis.
  • an "isolated polynucleotide or nucleic acid” is a polynucleotide or nucleic acid removed from its original environment (e.g., the natural environment if naturally occurring) and thus, altered by the "hand of man” from its natural state.
  • the term therefore covers, for example, (a) a DNA fragment of a naturally occurring genomic DNA molecule free of the coding sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA in the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein.
  • nucleic acids present in random, uncharacterized mixtures of different DNA molecules, transfected cells, or cell clones,
  • a naturally-occurring nucleic acid can be derived from mammals including mice, rats, and humans.
  • the known human sFlt-1 gene (Kendall RL et al. 1993 supra, GenBank accession number U01134), as shown in SEQ ID NO: 1, and mouse sFlt-1 gene (GenBank accession number D88690), as shown in SEQ ID NO: 3, can be used.
  • An sFlt-1 gene used in this invention can be synthesized based on its known sequence. For example, it is possible to clone the cDNA of sFlt-1 by performing a RT-PCR reaction on mRNA derived from a suitable source using a suitable DNA portion as a PCR primer. Such cloning can easily be performed by a person skilled in the art according to a reference, such as Maniatis T. et al., Molecular Cloning 2nd Ed., Cold Spring Harbor Laboratory Press (1989).
  • the sFlt-1 gene may have a partial deletion, substitution, or insertion of one or more nucleic acid, or may have other nucleotide sequence ligated therewith at the 5' terminus and/or 3' terminus thereof.
  • sFlt-1 activity means that the activity to bind to VEGF but not to result in signal transduction.
  • the phrase "functionally equivalent” means that the subject polypeptide retains a biologically significant activity that is characteristic of sFlt-1.
  • biologically significant activities of sFlt-1 include VEGF inhibitory activities that inhibit inflammation and migration of vascular smooth muscle cells.
  • the present invention includes polynucleotides comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 1, in which one or more amino acids are substituted, deleted, inserted and/or added, so long as the resulting protein retains sFlt-1 activity.
  • the present invention also includes polynucleotides that hybridize under stringent conditions with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1, so long as the resulting polynucleotide encodes a protein that are functionally equivalent to sFlt-1.
  • the determination of sFlt-1 can be conducted by methods well known to those skilled in the art, such as VEGF-binding assay as described in Duan, D-S, R. et al, (1991) J.BioLChem., 266, pp.413-418, and mitogen inhibition assay as described in WO94/21679.
  • Polynucleotides of the present invention can be obtained by methods well known to those skilled in the art. Examples of such methods include site-directed mutagenesis (Kramer, W and Fritz, HJ (1987) Methods in Enzymol. 154:350-367), hybridization technique (E.M. Southern, J. Mol. Biol. 1975, 98: 503-517) and polymerase chain reaction (PCR) technique (R.K Saiki et al, Science 1985, 230: 1350-1354; R.K. Saiki et al., Science 1988, 239: 487-491).
  • site-directed mutagenesis Kramer, W and Fritz, HJ (1987) Methods in Enzymol. 154:350-367
  • hybridization technique E.M. Southern, J. Mol. Biol. 1975, 98: 503-517
  • PCR polymerase chain reaction
  • those skilled in the art can generally isolate polynucleotides highly homologous to the polynucleotide shown in SEQ ID NO: 1 from other animals, using the polynucleotide shown in SEQ ID NO: 1 or a part thereof as probes or using the oligonucleotide which specifically hybridizes with the polynucleotide shown in SEQ ID NO: 1 as primers.
  • polynucleotides that can be isolated by hybridization techniques or PCR techniques and that hybridize with polynucleotides shown in SEQ ID NO: 1 are also included in the polynucleotides of the present invention. Examples of such polynucleotides include polynucleotides disclosed in WO94/21679.
  • Hybridization reactions to isolate polynucleotides as described above are preferably conducted under stringent conditions.
  • Hybridization may be performed with buffers that permit the formation of a hybridization complex between nucleic acid sequences that contain some mismatches. At high stringency, hybridization complexes will remain stable only where the nucleic acid molecules are almost completely complementary.
  • stringency may be increased by reducing the concentration of salt or by raising the hybridization temperature. Temperature conditions for hybridization and washing greatly influence stringency and can be adjusted using melting temperature (Tm).
  • Tm varies with the ratio of constitutive nucleotides in the hybridizing base pairs, and with the composition of the hybridization solution (concentrations of salts, formamide and sodium dodecyl sulfate).
  • concentration of salts, formamide and sodium dodecyl sulfate concentration of salts, formamide and sodium dodecyl sulfate.
  • an organic solvent such as formarnide
  • stringent hybridization conditions includes conditions comprising: ⁇ S'C, 2 x SSC, 0,1% SDS and those having a stringency equivalent to the conditions. In general, the higher the temperature, the higher is the homology between two strands hybridizing at equilibrium.
  • Polynucleotides isolated under higher stringency conditions are expected to encode a polypeptide having a higher homology at the amino acid level to the amino acid sequence shown in SEQ ID NO: 2.
  • high homology means an identity of at least 65% or more, more preferably 70% or more, still more preferably 80%, further more preferably 90% or more, and most preferably 95% or more, in the whole amino acid sequence.
  • An sFlt-1 protein encoded by the nucleic acids as described above includes human sFlt-1 as shown in SEQ ID NO: 2, mouse sFlt-1 as shown in SEQ ID NO: 4, and its variants.
  • the variants are preferably encoded by the nucleotide sequence having at least 65% identity to human sFlt-1 gene. More preferably, the variant is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, identical to the nucleotide sequence of human sFlt-1 gene.
  • an isolated polynucleotide of the present invention e.g., SEQ ID NO: 1
  • the comparison is made with the full length of the inventive sequence.
  • the isolated polynucleotide of the present invention is shorter than the prior art sequence, the comparison is made to a segment of the prior art sequence of the same length as that of the inventive sequence (excluding any loop required by the homology calculation).
  • the determination of percent identity between two sequences can be accomplished using any conventional mathematical algorithm, such as the BLAST algorithm by Karlin and Altschul (S. Karlin and S.F. Altschul, Proc. Natl. Acad. Sci. USA. 1990, 87: 2264-2268; S. Karlin and S.F. Altschul, Proc. Natl. Acad. Sci. USA. 1993, 90: 5873-5877).
  • the BLAST algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (S.F. Altschul et al., J. Mol. Biol. 1990, 215: 403).
  • Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389.
  • PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST are preferably used.
  • Polypeptides having amino acid sequences modified by deleting, adding and/or replacing one or more amino acid residues of a certain amino acid sequence have been known to retain the original biological activity (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, M. J. & Smith, M., Nucleic Acids Research (1982) 10, 6487-6500, Wang, A. et al., Science 224, 1431-1433, Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413).
  • the number of amino acids that are mutated by substitution, deletion, addition, and/or insertion is not particularly restricted. Normally, it is 10% or less, preferably 5% or less, and more preferably 1% or less of the total amino acid residues.
  • Amino acid substitutions may be made at one or more predicted, preferably nonessential amino acid residues.
  • a "nonessential" amino acid residue is a residue that can be altered from the wild-type sequence of a protein (e.g., the sequence shown in SEQ ID NO: 2) without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • An amino acid is preferably substituted for a different amino acid(s) that allows the properties of the amino acid side-chain to be conserved.
  • a “conservative amino acid substitution” is a replacement in which the amino acid residue is replaced with an amino acid residue having a chemically similar side chain. Groups of amino acid residues having similar side chains have been defined in the art.
  • These groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • An sFlt-1 gene of the present invention may be incorporated into various vectors. Any vectors can be used so long as they permit the in vivo expression of the gene. Examples of vectors includes plasmids, liposomes, and viral vectors. As plasmid vectors, eukaryotic plasmids are preferably used, including pCAGGS (Gene
  • a gene of this invention can be operably linked to promoter/enhancer elements.
  • the promoter/enhancer elements may be selected to optimize for the in vivo expression of the gene.
  • the promoter may be inducible or constitutive, and, optionally, tissue-specific. Promoters isolated from the genome of viruses that grow in mammalian cells, such as vaccinia virus 7.5K, SV40, HS V, adenoviruses MLP, MMTV, LTR and CMV promoters, may be used.
  • an sFlt-1 gene of the present invention can be encapsulated into any known liposome made of lipid bilayer such as an electrostatic liposome.
  • a liposome containing an sFlt-1 gene of the present invention can be fused to viruses such as inactivated Sendai virus (Hemagglutinating virus of Japan: HVJ).
  • the HVJ-liposome has very high fusing activity with the cell membrane as compared to the conventional liposomes.
  • the Z strain available from ATCC
  • HVJ strains for example, ATCC VR-907 and ATCC VR-105
  • Viral vectors can also be used.
  • Viral vectors can be DNA viruses or RNA viruses.
  • the viral vectors include detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Hemagglutinating virus of Japan envelope (HVJ-E, Sendai virus), SV40, and human immunodeficiency virus (HIV).
  • HVJ-E Hemagglutinating virus of Japan envelope
  • SV40 Sendai virus
  • HV human immunodeficiency virus
  • HV human immunodeficiency virus
  • an adenovirus vector system can be preferably used.
  • a vector carrying an sFlt-1 gene of the present invention can be formulated into an appropriate gene therapy agent.
  • gene therapy agent used herein means a pharmaceutical composition used as a dosage form for gene therapy.
  • the composition may vary depending on administration regimens described below (e.g. liquids).
  • an injection may be prepared by dissolving the gene into an appropriate solvent (a buffer such as PBS, physiological saline, sterile water, etc.).
  • the injection liquid may then be filter-sterilized with filter as needed and then filled into sterilized containers. Conventional carriers and so on may be added to the injection.
  • Liposomes such as HVJ-liposome, may take the form of suspensions, frozen formulations, centrifugation-concentrated frozen formulations, and the like.
  • Gene therapy agents of the present invention comprise a vector carrying an sFlt-1 gene, so that sFlt-1 is expressed in an amount effective to inhibit or treat inflammation of vessel walls or to inhibit formation of NIH.
  • Such inhibitory effects can be determined as described in the examples below.
  • inflammation-inhibitory effects can be determined by measuring the number of Mac3-positive monocytes (see Examples 2, 3, and 4, Figure 3E).
  • NIH formation-inhibitory effects can be determined by measuring intimal area, intima/media ratio, and % stenosis (see Example 4, Figure 3B, C, and D).
  • VEGF vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • VEGFiss and VEGF 16 or their corresponding isoforms should be determined.
  • the expression level can be measured for an mRNA level or protein level using a known method such as Northern blotting and Western blotting (e.g., Maniatis T. et al., supra).
  • NIH can be measured by cardio angiography (CAG) or intravascular ultrasounds (IVUS).
  • CAG cardio angiography
  • IVUS intravascular ultrasounds
  • a gene therapy agent of the present invention may be introduced into target cells or tissues of patients by in vivo methods or ex vivo methods.
  • In vivo methods permit direct introduction of the gene therapy agent into the body.
  • ex vivo methods certain cells are removed from human, the gene therapy agent is introduced into the cells, and the resulting cells are returned into the body thereafter (Nikkei Science, April 1994 issue pp.20-24; Monthly Yakuji, 36(1): 23-48 (1994); Supplement To Experimental Medicine 12(15) (1994); Handbook for Development and Research of Gene Therapy, NTS (1999)).
  • in vivo methods are preferred.
  • Illustrative methods of gene transfer into cells include the lipofection method, calcium phosphate co-precipitation method, DEAE-dextran method, direct DNA introduction methods using micro glass tubes, and the like.
  • Exemplary methods of gene transfer into tissues include internal type liposome method, electrostatic type liposome method, HVJ-liposome method, improved HVJ-liposome method (HVJ-AVE hposome method), receptor-mediated gene introduction, particle gun method, naked-DNA method, method of introduction with positively-charged polymers, etc.
  • electroporation may be applied following the gene transfer.
  • Microinjection or viral vectors can also be used for efficient gene transfer or transgene expression.
  • a gene therapy agent of this invention can be administered parenterally, preferably intramuscularly, to the site of vascular injury.
  • a dosage of an agent of this invention varies depending on the age, gender, and symptoms of the patient, but sFlt-1 gene can be administered at a dose of about 0.0001 mg to about lOOmg, preferably about 0.001 to about 10 mg per day per adult patient.
  • a gene of this invention can be administered in a range of about 1 to about 4000 ⁇ g, preferably about 10 to about 400 ⁇ g per adult patient.
  • the therapeutic agent of this invention may be administered once every few days or every few weeks, or once per week. Frequency of administration is to be selected depending on the symptoms of the patients. In compliance with the object of the treatment, plural administration is suitable.
  • the gene therapy of the present invention is effective for inhibiting inflammation of vessel wall and/or formation of NIH. Inflammation of vessel wall and formation of NIH are symptoms observed after vascular injury caused by post coronary intervention restenosis, atherosclerosis, or arteriosclerosis.
  • the gene therapy of the present invention can be applied to patients with risk of these diseases. The risk factors for these diseases include hypercholesterolemia.
  • Figure 1 shows VEGF expression in cuffed femoral artery.
  • A A photograph showing time course of VEGF mRNA levels. Expression of arterial VEGF and ⁇ -actin mRNA after cuff placement. mRNA levels were assessed at the indicated times. This is a representative assay from five separate experiments.
  • FIG. 1 Photographs showing cross-sections of intact or cuffed femoral arteries were stained immunohistochemically against VEGF, VEGF receptor 1 (Flt-1), VEGF receptor 2 (Flk-1) or vWF 7 or 21 days after cuff placement. Bar indicates 50 ⁇ m.
  • Figure 2 shows immunofluorescence staining of VEGF receptors, monocytes, and ⁇ -SM actin in cuffed femoral artery.
  • A Micrographs of cuffed femoral arteries stained with Flt-1 (VEGF-R1, green) and ⁇ -SM actin (red), with Flk-1 (VEGF-R2, green) and ⁇ -SM actin (red), and with Mac-3 (green) and Flt-1 (VEGF-R1, red) 7 days after cuff placement. Bar indicates 10 ⁇ m.
  • B Micrographs of cuffed femoral arteries stained with Flt-1 (VEGF-R1) and ⁇ -SM actin, and Flk-1 (VEGF-R2) and ⁇ -SM actin in the cuffed femoral arteries 21 days after cuff placement. Single fluorescence-positive cells were stained green or red, whereas double-positive cells were stained yellow. Scale bar indicates 10 ⁇ m.
  • Figure 3 shows histopathology of cuffed femoral artery.
  • A Photographs showing time course of cuff injury-induced NIH and effect of sFlt-1 gene transfer. Micrographs of cross-sections of control (intact) and cuffed arteries stained with van Gieson Elastica (vGE) on days 3, 7, and 21 are shown. Scale bar indicates 100 ⁇ m.
  • B, C, and D Effects of sFlt-1 gene transfer on neointimal thickening (B), intima/media ratio (C), % stenosis (D) 21 days after cuff placement.
  • E Effects of sFlt-1 gene transfer on inflammatory and proliferative changes 7 days after cuff placement.
  • Figure 4 shows contribution of bone marrow-derived cells in the development of the neointima after cuff placement.
  • FIG. 5 is a photograph showing effects of sFlt-1 gene transfer on chemokines and chemokine receptors (MCP-1, CCR2, RANTES, CCR1, MIP-1 ⁇ , CXCR2, eotaxin, MIP-2), adhesion molecules (ICAM-1, VCAM-1), cytokines (IL-6,TGF- ⁇ ), VEGF, and Flt-1 in cuffed femoral arteries.
  • MCP-1 chemokines and chemokine receptors
  • IAM-1 adhesion molecules
  • VCAM-1 VCAM-1
  • cytokines IL-6,TGF- ⁇
  • VEGF Flt-1 in cuffed femoral arteries.
  • Data are expressed as the ratio of each mRNA to the corresponding GAPDH mRNA. *P ⁇ 0.01 versus control site; fP ⁇ 0.01 versus empty plasmid group; NS indicates not significant.
  • N 5-6.
  • the 3.3-kb mouse sFlt-1 gene (Genbank accession number D88690; nucleotide and amino acid sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively) was obtained from a mouse lung cDNA library (Kondo K, Hiratsuka S, Subbalakshmi E, Matsushime H, Shibuya M. Genomic organization of the flt-1 gene encoding for vascular endothelial growth factor (VEGF) receptor- 1 suggests an intimate evolutionary relationship between the 7-Ig and the 5-Ig tyrosine kinase receptors. Gene.
  • VEGF vascular endothelial growth factor
  • Apolipoprotein E knockout mice (8-10 week old) with a genetic background of C57BL/6J were purchased from Jackson Laboratory (Bar Harbor, ME) and fed with commercial standard chow. Placement of cuff and gene transfer were performed as previously described (Zhao Q et al., supra; and Egashira K et al., supra).
  • a non-constrictive polyethylene cuff (1.5-mm ng; PE20, 0.38-mm inner diameter, 1.09-mm outer diameter) was placed loosely around the left femoral artery.
  • Either empty plasmid or sFlt-1 plasmid 300 ⁇ g/100 ⁇ l PBS was injected into the right femoral muscle using a 27-gauge needle immediately after and 10 days thereafter. To enhance transgene expression, these animals received electroporation at the injected site immediately after injection.
  • Six electric pulses of 100 V for 50 ms were applied with the use of an electric pulse generator CUY21 (BTX).
  • Cuff placement was also performed in wild-type mice with a genetic background of C57BL/6J whose bone marrow was replaced with that of ROSA26 mice, which ubiquitously expresses ⁇ -galactosidase (LacZ) (Sata M et al, supra).
  • LacZ ⁇ -galactosidase
  • Lethally-irradiated wild-type mice received lxlO 6 bone-marrow cells from a ROSA26 mouse.
  • a cuff was placed around the left femoral artery. The femoral artery was excised and stained with X-gal solution for 7 h, then further fixed in 4% paraformaldehyde.
  • mice were anesthetized with pentobarbital, and the femoral artery was harvested, fixed overnight in 3.7% formaldehyde in phosphate buffered saline, and paraffin-embedded (Egashira).
  • Indirect immunofluorescence double-staining with matched primary and fluorescein conjugated secondary antibodies was used to stain for colocalization with VEGF receptors in smooth muscle cells or monocytes: Rabbit anti- mouse Flt-1 (Santa Cruz Biotech), rabbit anti-mouse Flk-1 (Santa Cruz Biotech), rat anti-mouse Mac-3, anti-smooth muscle actin ( ⁇ -SMA) (Boehringer Mannheim), anti-rabbit IgG conjugated with FITC or rhodamine, and anti-rat IgG conjugated with FITC or rhodamine (Santa Cruz Biotech).
  • PCR polymerase chain reaction
  • VEGF vascular endothelial growth factor
  • Primers used for amplification of VEGF were 5'-GGA TCC ATG AAC TTT CTG CT-3' (SEQ ID NO: 5) and 5'-GAA TTC ACC GCC TCG GCT TGT C-3' (SEQ ID NO: 6) with expected sizes of 654 bp, 582 bp, and 450 bp for the three VEGF isoforms (VEGF 188, 164, and 121, respectively).
  • Primers for the internal control , ⁇ -actin were 5'-ATG GAT GAC GAT ATC GCT-3' (SEQ ID NO: 7) and 5'-ATG AGG TAG TCT GCT AGG T-3' (SEQ ID NO: 8) with an expected product of 550 bp. PCR products were separated by 2% agarose gel electrophoresis, visualized using ethidium bromide, photographed, and analyzed by scanning densitometry.
  • RNAse protection assays were performed using 5 ⁇ g of total RNA with two custom template sets according to the manufacturer's protocol (PharMingen). After RNAse digestion, protected probes were resolved on denaturing polyacrylamide gels and quantified using a BASS-3000 system (Fuji Film). The value of each hybridized probe was normalized to that of the internal controls, L32 and GAPDH, included within each template set.
  • Statistical Analysis were performed using 5 ⁇ g of total RNA with two custom template sets according to the manufacturer's protocol (PharMingen). After RNAse digestion, protected probes were resolved on denaturing polyacrylamide gels and quantified using a BASS-3000 system (Fuji Film). The value of each hybridized probe was normalized to that of the internal controls, L32 and GAPDH, included within each template set. Statistical Analysis
  • Example 1 Plasma lipid levels Plasma total cholesterol, triacylglycerol, and high density lipoprotein-cholesterol levels were determined with commercially available kits (Wako Pure Chemicals, Osaka, Japan). There were no statistically significant differences in serum total cholesterol and triacylglycerol levels among the three groups; the control group, the empty plasmid group, and the sFlt-1 group. Total cholesterol and triacylglycerol levels were 503 ⁇ 11 and 38 ⁇ 6 mg/dL in the control group, 512 ⁇ 16 and 40 ⁇ 5 mg/dL in the empty plasmid group, and 497 ⁇ 10 and 39 ⁇ 3 mg/dL in the sFlt-1 group.
  • VEGF protein 100 ng/mL was injected subcutaneously into the flanks of C57BL/6J mice. The matrigels were then removed 7 or 14 days after injection, and angiogenesis and inflammation were examined by histopathologic analysis.
  • Example 3 Increased expression of VEGF mRNA and immunoreactivity
  • VEGF 121 mRNA was undetectable before and after cuff placement.
  • Immunohistochemical staining indicated that compared to faint staining in the control artery, VEGF increased in the vicinity of inflammatory lesions (mononuclear cell infiltration) in the intima and adventitia on day 7 and in cells of three layers of cuffed artery on day 21 (Figure 1C).
  • the endothelial layer, as detected by vWF staining, was preserved before and after cuff placement ( Figure 1C).
  • Flt-1 was undetectable except in endothelial layers in control intact arteries, but was drastically increased in the intima, media, and adventitia 7 and 21 days after cuff placement (Figure 1C).
  • VEGFR-2 (Flk-1) was not increased on day 7, but did increase on day 21.
  • mice were killed on days 1, 3, 7, 14 , and 21.
  • the present inventor used bone marrow-transplanted mice whose bone marrow expressed ⁇ -galactosidase.
  • ⁇ -galactosidase (LacZ) of normal and cuffed artery was stained 21 days after cuff placement. LacZ-positive cells were rarely observed in the neointima or media ( Figure 4). Some mononuclear leukocytes recruited into in the adventitia were positive for LacZ.
  • sFlt-1 gene transfer significantly reduced NIH (increases in neointimal area, intima/media ratio, and luminal stenosis) 21 days after cuff placement ( Figure 3A, B,C, and D).
  • RNAse protection assays 7 days after cuff placement were examined by RNAse protection assays 7 days after cuff placement (Figure 5). Gene expression was upregulated after cuff placement. sFlt-1 gene transfer did not affect gene expression of RANTES, MlP-l ⁇ , TGF- ⁇ , MIP-2, but prevented or attenuated the increased gene expression of CCRl, IL-6, CCR2, MCP-1, Flt-1, CXCR2, eotaxin, VCAM-1, ICAM-1, and VEGF.
  • VEGF by sFlt-1 gene transfer can be an attractive anti-VEGF therapy for inflammatory vascular diseases and other inflammatory disorders.
  • this gene therapy is useful to inhibit the development of NIH after vascular injury caused by post coronary intervention restenosis, atherosclerosis, arteriosclerosis, or edema. Therefore ⁇ the compositions and methods of the present invention can be applied to a patient with risk of these diseases, including a patient with hypercholesterolemia.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Obesity (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Diabetes (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Urology & Nephrology (AREA)
  • Vascular Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des compositions et des procédés permettant d'inhiber l'inflammation de la paroi vasculaire et/ou la formation d'hyperplasie de néo-intima par thérapie génique au moyen d'un gène Flt-1(sFlt-1) soluble. VEGF possède un rôle essentiel dans le développement de l'hyperplasie de néo-intima en provoquant une inflammation. Le transfert du gène sFlt-1 sur le site de lésions vasculaires bloque la transduction de signaux VEGF à médiation de Flt-1, ce qui permet d'inhiber l'inflammation précoce ainsi que l'hyperplasie de néo-intima tardive. Cette invention sert à inhiber ou traiter l'inflammation de parois vasculaires et/ou la formation d'hyperplasie de néo-intima chez un patient qui présente des risques de resténose, d'athérosclérose, d'artériosclérose ou d'oedème après une intervention coronarienne.
EP04717875A 2003-03-07 2004-03-05 Compositions et procedes permettant d'inhiber l'inflammation des parois vasculaires et la formation d'hyperplasie de neo-intima Withdrawn EP1601696A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US45272003P 2003-03-07 2003-03-07
US452720P 2003-03-07
PCT/JP2004/002930 WO2004078786A1 (fr) 2003-03-07 2004-03-05 Compositions et procedes permettant d'inhiber l'inflammation des parois vasculaires et la formation d'hyperplasie de neo-intima

Publications (1)

Publication Number Publication Date
EP1601696A1 true EP1601696A1 (fr) 2005-12-07

Family

ID=32962744

Family Applications (1)

Application Number Title Priority Date Filing Date
EP04717875A Withdrawn EP1601696A1 (fr) 2003-03-07 2004-03-05 Compositions et procedes permettant d'inhiber l'inflammation des parois vasculaires et la formation d'hyperplasie de neo-intima

Country Status (3)

Country Link
EP (1) EP1601696A1 (fr)
JP (1) JP2007528355A (fr)
WO (1) WO2004078786A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080171033A1 (en) * 2004-08-26 2008-07-17 Chiwen Chang Compositions and methods for treating diseases associated with angiogenesis and inflammation

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001501471A (ja) * 1996-09-24 2001-02-06 メルク エンド カンパニー インコーポレーテッド 血管新生の阻害のための遺伝子治療
EP1297016B1 (fr) * 2000-05-12 2006-03-22 Vlaams Interuniversitair Instituut voor Biotechnologie vzw. Utilisation d'inhibiteurs du facteur de croissance placentaire pour le traitement de l'angiogenese pathologique, de l'arteriogenese pathologique, de l'inflammation, de la formation tumorale et/ou de la fuite vasculaire

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of WO2004078786A1 *
SHIBATA M. ET AL., HISTOCHEM CELL BIOL., vol. 116, 2001, pages 471 - 481 *

Also Published As

Publication number Publication date
JP2007528355A (ja) 2007-10-11
WO2004078786A1 (fr) 2004-09-16

Similar Documents

Publication Publication Date Title
Sprengel et al. Molecular cloning and expression of cDNA encoding a peripheral-type benzodiazepine receptor
EP0935001B1 (fr) Nouveaux facteurs analogues au vegf
JP4439490B2 (ja) 血管内皮細胞増殖因子２
Frieser et al. Cloning of the mouse laminin α4 cDNA. Expression in a subset of endothelium
EP0750628B1 (fr) Facteur-10 de croissance du fibroblaste
US7750133B2 (en) Vascular endothelial cell growth inhibitor, VEGI-192a
EP1313762B1 (fr) Genes suppresseurs
JP2004536579A (ja) 血管内皮増殖因子２
EP1021464A1 (fr) Recepteur cellulaire de la proteine virale (vpr) du vih-1 essentielle pour la transition de phase g2/m du cycle cellulaire
JP2010047588A (ja) 新規膜貫通蛋白質をコードする遺伝子
US20020119541A1 (en) Tumor suppressor CAR-1
JP2007527193A (ja) ケモカインに対する核受容体としてのｔｈａｐタンパク質と、転写調節、細胞増殖、および細胞分化におけるその役割
JPH1084976A (ja) 新規ヒトｇ−蛋白質結合レセプター
US20060234969A1 (en) Compositions and methods for inhibiting inflammation of vessel walls and formation of neointimal hyperplasia
EP1601696A1 (fr) Compositions et procedes permettant d'inhiber l'inflammation des parois vasculaires et la formation d'hyperplasie de neo-intima
CA2356009C (fr) Facteur de croissance endotheliale vasculaire- x
US6800454B2 (en) Nucleic acid molecule encoding chemokine-like factor 1 (CKLF1)
US6783953B1 (en) Vascular endothelial growth factor-X
WO1998033819A9 (fr) Recepteurs cellulaires des adenovirus du sous-groupe c et des virus coxsackie du groupe b
JP2005312304A (ja) 新規インターフェロン制御因子活性化ポリペプチド及びそれをコードする核酸
US6768003B1 (en) Nucleic acids that encode transcriptional adaptor proteins
US20050042655A1 (en) Novel hematopoietic regulatory factors and methods of use thereof
WO2000029578A1 (fr) Clonage et caractérisation de deux facteurs de transcription de l'arn messager
US6833237B1 (en) Genes involved in stroke response and/or regulated by FK506, proteins encoded thereby, and methods of use
AU2005200547A1 (en) Vascular Endothelial Growth Factor 2

Legal Events

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

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20050929

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20061024

17Q First examination report despatched

Effective date: 20061024

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

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

18D Application deemed to be withdrawn

Effective date: 20081118