WO2008088358A2 - Expression ex vivo et in vivo du gène thrombomoduline pour le traitement de maladies cardiovasculaires et vasculaires périphériques - Google Patents

Expression ex vivo et in vivo du gène thrombomoduline pour le traitement de maladies cardiovasculaires et vasculaires périphériques Download PDF

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WO2008088358A2
WO2008088358A2 PCT/US2007/006371 US2007006371W WO2008088358A2 WO 2008088358 A2 WO2008088358 A2 WO 2008088358A2 US 2007006371 W US2007006371 W US 2007006371W WO 2008088358 A2 WO2008088358 A2 WO 2008088358A2
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seq
promoter
gutless
vector
sequence
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PCT/US2007/006371
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WO2008088358A3 (fr
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Lakshman R. Sehgal
Jonathan Wong
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Biovec, Llc
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Priority claimed from US11/650,478 external-priority patent/US7501114B2/en
Priority claimed from US11/650,479 external-priority patent/US7481998B2/en
Priority claimed from US11/685,474 external-priority patent/US7803365B2/en
Application filed by Biovec, Llc filed Critical Biovec, Llc
Priority to EP07772782A priority Critical patent/EP2099438A4/fr
Priority to CA2674563A priority patent/CA2674563C/fr
Publication of WO2008088358A2 publication Critical patent/WO2008088358A2/fr
Publication of WO2008088358A3 publication Critical patent/WO2008088358A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/366—Thrombomodulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/41—Porphyrin- or corrin-ring-containing peptides
    • A61K38/42—Haemoglobins; Myoglobins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention is directed to methods and compositions for the treatment of cardiovascular and peripheral vascular diseases, and in particular, is directed to methods and compositions for ex vivo and in vivo expression of the thrombomodulin gene using gutless adenovirus vector.
  • Atherosclerosis is one of the chief causes of morbidity and mortality in the United States and many other countries of the world. (Zuckeroraun et al., Arch Surg. 137:854-861 [2002]; Kibbe et al., Circ Res. 86:829-33 [200O]). This process can result in limiting the flow of blood to the heart, kidneys and th'e peripheral vessels, to name a few.
  • Current approaches to the treatment of lesions in the arteries include coronary artery by-pass graft (CABG) surgery and angioplasty with or without the placement of a stent. The latter may serve as a vehicle for drug delivery, as is currently being tested in clinical trials.
  • CABG coronary artery by-pass graft
  • Cardiovascular diseases are the result of; complex pathophysiologic processes that involve the expression of many proteins and molecules that can adversely affect the grafted vessel (Shears et al., J. Am Coll Surg., 187(3):295-306 [1998]; Ross et al., Nature, 362:801-9 [1993]). Approximately 15- 30% of patients receiving vein grafts for coronary or peripheral vascular disease require follow-up treatment, either in the form of angioplasty or new grafts.
  • Thrombomodulin is an integral membrane glycoprotein expressed on the surface of endothelial cells (Sadler et al., Trhomb Haemost,, 78:392- 95 [1997]). It is a high affinity thrombin receptor that converts thrombin into a protein C activator. Activated protein C then functions as an anticoagulant by inactivating two regulatory proteins of the clotting system, namely factors Va and VI [I]a (Esmon et al., Faseb J., 9:946-55 [1995]). The latter two proteins are essential for the function of two of the coagulation proteases, namely factors IXa and Xa. TM thus plays an active role in blood clot formation in vivo and can function as a direct or indirect anticoagulant.
  • Nitric oxide synthase an enzyme expressed by endothelial cells has been shown in animal models to inhibit intimal hyperplasia, especially the inducible enzyme (iNOS) (Salmaa et al., Lancet, 353:1729-34 [1999]; Palmer et al., Nature, 327:524-26 [1987]; Kubes et al., PNAS USA., 88:4651-5 [1991]).
  • iNOS inducible enzyme
  • VEGF Vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • PDGF platelet derived growth factor
  • a gene therapy approach is currently under clinical! investigation. It involves the injection, directly into heart muscles, of an adenoviral vector delivery system containing the gene for the expression of vascular endothelial growth factor (VEGF). This is being tested in patients whose coronary vessels are not amenable to standard grafting procedures. However, some recent adverse clinical events demonstrated that injection of large quantities of adenovirus vectors is associated with significant risks. Accordingly, there still exists a need for a method to effectively introduce therapeutic genes, such as TM, into vascular tissues.
  • TM therapeutic genes
  • One aspect of the present invention relates to methods for treating a vascular disease in a mammal.
  • the method comprises the steps of: evacuating a clot from a blood vessel in the mammal, isolating a segment of the blood vessel around the evacuation site, and infecting the segment of blood vessel in vivo using a gutless adenoviral vector comprising a polynucleotide encoding a thrombomodulin protein or its variant, wherein the thrombomodulin protein or its variant is expressed in a amount sufficient to reduce re-occlusion [or intimal hyperplasia in the infected blood vessel, and wherein said gutless adenoviral vector comprises the nucleotide sequence of SEQ ID N0:13 or SEQ 1D:NO:15.
  • the method comprises thejstep of administering a therapeutically effective amount of a gutless adenovirus vector into a segment of a blood vessel in vivo using a stent, wherein the gutle'ss adenovirus vector comprises the nucleotide sequence of SEQ ID NO: 13 or SEQ FD 1 NO: 15, and is capable of expressing a thrombomodulin protein or a variant of the thrombomodulin protein.
  • the method comprises the 'step of administering intravenously an effective amount of a gutless adenoviral vector comprising a polynucleotide encoding a thrombomodulin protein or its variant, wherein the gutless adenoviral vector comprises the nucleotide sequence of SEQ ID NO: 13 or SEQ lD NO:15.
  • the polynucleotide encoding the thrombomodulin protein or its variant is under the control of a CMV promoter or an RSV promoter.
  • the polynucleotide encoding the thrombomodulin protein or its variant is under the control of a liver specific promoter selected from the group consisting of albumin promoter, alpha- l ⁇ antitrypsin promoter and alpha-fetoprotein promoter.
  • the gutless virus vector is administered through a portal vein.
  • Another aspect of the present invention pertains toja gutless adenovirus vector comprising a polynucleotide encoding a thrombomodulin protein having the amino acid sequence of SEQ ID NO:2, a regulatory element operably linked to the polynucleotide sequence; and a stuffer comprising the nucleotide 1 sequence of SEQ ID NO: 13 or SEQ ID NO: 15, wherein the regulatory element is a liver specific promoter.
  • the liver specific promoter is selected from the group consisting of albumin promoter, alpha-l -antitrypsin promoter and alpha- fetoprotein promoter.
  • the method comprises theisteps of infecting a segment of blood vessel in vitro using a gutless adenoviral vector comprising a polynucleotide encoding a thrombomodulin protein or its variant, and grafting the virus-treated blood vessel in the mammal, wherein the thrombomodulin protein or its variant is expressed in a amount sufficient to reduce re-occlusionior intimal hyperplasia in the grafted blood vessel, and wherein the gutless adenoviral vector comprises the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 15.
  • Another aspect of the present invention pertains to a gutless adenovirus vector comprising a polynucleotide encoding a thrombomodulin protein having the amino acid sequence of SEQ ID NO:2, a regulatory element operably linked to the polynucleotide sequence; and a stuffer comprising the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 15.
  • Yet another aspect of the present invention pertains
  • Figure 1 is a schematic drawing of an embodiment of the backbone shuttle vector pShuttie-ITR-HPRT.
  • Figure 2 is a schematic drawing of an embodiment of the full length backbone vector pTM-final
  • Figure 3 is a picture of a Western blot showing hTM expression in HEK 293 cells transfected with pTM-final (the full size backbone of gutless Ad.hTM). Lanes 1-3: lysate from control cells; Lanes 4-6, lysate from pTM-final transfected cells.
  • Figure 4 is a picture of a Western slot blot showing hTM expression in 293FLP cells (passage number 2 (P2) during viral amplification). Row 1, lane 1-3: TM detection using 5ul cell lysate of P2. Row 2, lane 1-3: TM detection using 30ul cell lysate of P2. Row 3, lane 1-3: negative control cells.
  • Figure 5 is a picture of a Western blot showing hTM expression in rat vena cava infected with gutless TM virus.
  • Figure 6 is a picture of a Western bolt showing TM expression in CRE cells at passage number 1-6 (P1-P6).
  • Figure 7 is a composite of images showing gutless adenovirus- mediated luciferase expression in rat tail vein.
  • Figure 8 is a diagram showing TM expression in livers of non-infected rats (con) and TM gutless virus infected rats (TM virus).
  • Figure 9 is a picture of Western blots using a anti-TM antibody (blot 1 ) and plasma from animals infected with TM virus (blots 2-4).
  • the practice of the present invention will employ, unless otherwise indicated, conventional methods of histology, virology, microbiolqgy, immunology, and molecular biology within the skill of the art. Such techniques are explained fully in the literature. AU publications, patents and patent applications c'ited herein, whether supra or infra, are hereby incorporated by reference in their entirety . [030]
  • the primary object of the present invention is to provide methods for treating vascular diseases using gene delivery technologies.
  • One aspect of the present invention relates to a method for treating a vascular disease by introducing a DNA sequence encoding a TM protein or its variant into a segment of aiblood vessel in vitro using a gutless adenovirus vector and grafting the virus-treated vessel in a patient affected by a vascular disease.
  • the virus-mediated TM expression reduces re- occlusion and intimal hyperplasia in the grafted vessel. This ex vivo approach eliminates the need to inject a large quantity of virus into a patient and hence significantly reduces the viral-related toxicity.
  • the method is used for a coronary artery bypass. In another embodiment, the method is used for the treatment of peripheral vascular diseases. In yet another embodiment, the method is used for the maintenance of vein access in renal dialysis patients.
  • Another object of the present invention is to provide a method to deliver a gutless adenovirus vector carrying a DNA sequence encoding a TM protein or its variant using a stent.
  • the viral vector is embedded in the stent and is released only at a treatment site. Since the viral infection is restricted at the treatment site and the surrounding area, only a small amount of the virus is needed and the virus-related toxicity is reduced.
  • Yet another object of the present invention pertains, to a gutless adenovirus carrying a TM gene.
  • the gutless adenovirus which contains a regulatory element operably linked to a DNA sequence ⁇ encoding a TM protein or its variant and a polyA sequence, is produced using a novel shuttle vector containing a pBR322 replication origin, a selection marker, an adenovirus left inverted terminal repeat, an adenovirus encapsidation signal, a stuffer sequence, and an adenovirus left inverted terminal repeat.
  • the regulatory element is a constitutive promoter such a CMV promoter and RSV promoter. In another embodiment, the regulatory element is an inducible promoter.
  • the forth object of the present invention is to provide a pharmaceutical composition which comprises an effective amount of gutless adenovirus carrying a TM gene of the present invention and a pharmaceutically acceptable carrier.
  • Such compositions may be liquids or lyophilized or otherwise dried formulations and may further include diluents of various buffer content, (e.g., Tris-HCl, 1 acetate, phosphate) pH and ionic strength, additives such as albumin and gelatin to prjevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g., glycerol, polyethylene glycerol); anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g. Thimerosal, benzyl alcohol, parabens).
  • buffer content e.g., Tris-HCl, 1 acetate, phosphate
  • Gene transfer or “gene delivery” refers to methods or systems for reliably introducing a particular nucleotide sequence (e.g., DNA) into targeted cells, The introduced nucleotide sequences may persist in vivo in episorhal forms or integrate into the genome of the target cells.
  • Gene transfer provid'es a unique approach for the treatment of acquired and inherited diseases, andia number of systems have been developed in the art for gene transfer into mammalian cells. See, e.g., U.S. Pat. No. 5,399,346.
  • the term "effective amount" refers ,to a level of infection which brings about at least partially a desired therapeutic or prophylactic effect in an organ or tissue infected by the method of the present invention.
  • the infection with an effective amount of the vector carrying genetic material of interest can then result in the modification of the cellular activities, e.g., a.change in phenotype, in an organ or a tissue that has been infected by the method of the present invention.
  • the infection with an effective amount of the vector carrying genetic material of interest results in modulation of cellular activity in a significant number of cells of an infected organ or a tissue.
  • a gene transfer "vector” refers to any agent, such as a plasmid, phage, transposon, cosmid, chromosome, liposome, DNA-viral conjugates, RNA/DNA oligonucleotides, virus, bacteria, etc., which is capable of transferi]ing gene sequences into cells.
  • the term includes cloning and expression vehicles including "naked" expression vectors, as well as viral and non-viral vectors.
  • a vecto ⁇ may be targeted to specific cells by linking a target molecule to the vector.
  • a targeting molecule is any agent that is specific for a cell or tissue type of interest, including for example, a ligand, antibody, sugar, receptor, or other binding molecule.
  • the invention is also intended to include such other forms of vectors which serve equivalent functions and which become known in the art subsequently hereto.
  • control element refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
  • promoter is used herein in its ordinary sense to refer to a, DNA regulatory sequence that is sufficient for RNA polymerase recognition, binding and transcription initiation. Additionally, a promoter includes sequences that modulate the recognition, binding and transcription initiation activity of RNA polymerase. Such sequences may be cis acting or may be responsive to trans acting factors. Depending upon the nature of the regulation, promoters may be constitutive, tissue specific, or regulated. Examples of constitutive promoters include, but are not limited to, SP6, T4, Tl, SV40 early promoter, cytomegalovirus (CIvIV) promoter, RSV promoter, and Moloney murine leukemia virus (MMLV) promoter.
  • CIvIV cytomegalovirus
  • MMLV Moloney murine leukemia virus
  • tissue specific promoters include, but are not limited to, liver specific promoters such as albumin promoter, alpha 1 -antitrypsin promoter and alpha-fetoprbtein promoter, and muscle specific promoters such as muscle creatine kinase (MCK) promoter, myosin promoter, and ⁇ -actin promoter.
  • MCK muscle creatine kinase
  • the term "transduction" denotes the delivery of a DNA molecule to a recipient cell either in vivo or in vitro, via a replication-defective viral vector, such as via a recombinant adenovirus.
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • the control elements need not be contiguous with the coding sequence, so long as the function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • primer refers to an oligonucleotide which is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated.
  • An oligonucleotide "primer” may occur naturally, as in a purified restriction digest or may be produced synthetically.
  • a primer is selected to be "substantially" complementary to a strand of specific sequence of the template.
  • a primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur.
  • a primer sequence need not reflect the exact sequence of the template. For example,ja non- i complementary nucleotide fragment may be attached to the 5' end] of the primer, with the remainder of the primer sequence being substantially complementary to the strand.
  • Non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity
  • Hybridization methods involve the annealing of a complementary sequence to the target nucleic acid (the sequence to be detected). The ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interaction is a well-recognized phenomenon.
  • the initial observations of the "hybridization” process by Marmur and LanQ,>PNAS USA 46:453 (1960) and Doty et al., PNAS USA 46:461 (1960) have been followed by the refinement of this process into an essential tool of modern biology.
  • nucleic acid sequence refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3 1 end of the other, is in "a ⁇ tiparallel association.”
  • Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention and include, for example, inosine and 7-deazaguanine, Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases.
  • nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs.
  • Tm melting temperature
  • Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by'comparing the sequences using standard software available in sequence data ban'ds, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system.
  • Suitable conditions include those characterized by a hybridization buffer comprising 0.9M sodium citrate ("SSC") buffer at a temperature of about 37°C and washing in SSC buffer at a temperature of about 37 0 C; and preferably in a hybridization buffer comprising 20% formamide in 0.9M SSC buffer at a temperature of about 42 0 C and washing in 0.2x SSC buffer at about 42°C.
  • Stringency conditions can be further varied by modifying the temperature and/or salt content of the buffer, or by modifying the length of the hybridization probe as is known to those of skill in the art. Defining appropriate hybridization conditions is within the skill of the art. See e.g., Sambrook, J. Fritsch, E. J., & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, NY).
  • probe refers to a labeled oligonucleotide which forms a duplex structure with a sequence in another nucleic acid, due to complementarity of at least one sequence in the probe with a sequence in the other nucleic acid.
  • label refers to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like.
  • Oligonucleotide primers matching or complementary to a gene sequence refers to oligonucleotide primers capable of facilitating the template- dependent synthesis of single or double-stranded nucleic acids. Oligonucleotide primers matching or complementary to a gene sequence may be used in PCRs, RT- PCRs and the like.
  • a "consensus gene sequence” refers to a gene sequence which is derived by comparison of two or more gene sequences and which' describes the nucleotides most often present in a given segment of the genes; the consensus sequence is the canonical sequence.
  • thrombomodulin refers to both the natural protein and soluble peptides having the same characteristic biological activity of membrane- bound or detergent solubilized (natural) thrombomodulin. These, soluble peptides are also referred to as "wild-type” or “non-mutant” analog peptides. .
  • Biological activity is the ability to act as a receptor for thrombin, increase the activation of protein C, or other biological activity associated with native thrombomodulin.
  • Oxidation resistant TM analogs are these soluble peptides that in addition to being soluble contain a specific artificially induced mutation in their amino acid sequence.
  • thrombomodulin variant is a polypeptide that differs from a native thrombomodulin polypeptide in one or more substitutions ⁇ deletions, additions and/or insertions, such that the bioactivity of the native thrombomodulin polypeptide is not substantially diminished or enhanced.
  • the bioactivity of a thrombomodulin variant may be enhanced or diminished by, less than 50%, and preferably less than 20%, relative to the native protein.
  • Preferred variants include those in which one or more portions, such as an N-terminal leader sequence or transmembrane domain, have been removed.
  • Other preferred variants include variants in which a small portion (e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removed from the - and/or C-terminal of the mature protein..
  • a thrombomodulin variant contains conservative substitutions.
  • a "conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such' that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
  • Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of th'e residues.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and ar ⁇ ino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.
  • a variant may also, or alternatively, contain nonconservative changes.
  • variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer.
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the bioattivity, secondary structure and hydropathic nature of the polypeptide.
  • Thrombomodulin variants preferably exhibit at least about 70%, more preferably at least about 90% and most preferably at least about 95% sequence homology to the original thrombomodulin polypeptide.
  • a thrombomodulin variant also includes a thrombomodulin polypeptides that is modified from the original thrombomodulin polypeptides by either natural processes, such as posttranslatio ⁇ al processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as welljas in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications.
  • Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or withoutlbranching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross links, formation of cysteine, formation of pyroglutamate, formulation, gammacarboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation,' oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • Adenovirus vectors Adeno
  • adenovirus The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lyric viral life cycle (Curie DT, Ann N Y Acad Sc i 886, 158-171 [1991]).
  • Suitable adenoidal vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art.
  • Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium, endothelial cells and muscle cells. Additionally, introduced adenoidal DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoidal genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery ve'ctors (Haj-Ahmand et al. J.
  • Adenovirus vectors have been successfully tested in a number of animal models (Ragot et al. Nature 361, 647-650 [1993]; Howell et al. Hum Gene Ther 9, 629-634 [1998]). Nonetheless, the toxicity and immunoge.nicity remain major hurdles to overcome before the adenovirus vectors can be safely used in humans.
  • Ad Adenoviruses
  • the adenovirus genome is complex and contains over 50 open reading frames (ORFs). These ORFs are overlapping and genes encoding one protein are often embedded within genes coding for other Ad proteins.
  • Expression of Ad genes is divided into an early and a late phase.
  • the early genes comprise EIa, EIb, E2a, E2b, E3 and E4, which are transcribed prior to replication of the viral genome.
  • the late genes e.g., Ll-5) are transcribed after replication of the viral genome.
  • the products of the late genes are predominantly components of the virion, as well as proteins involved in the assembly of virions.
  • the so-called “gutless” rAd vectors contain a minimal amount of adenovirus DNA and are incapable of expressing any adenovirus antigens (hence the term "gutless”).
  • the gutless rAd vectors provide the significant advantage of accommodating large inserts of foreign DNA while completely eliminating the problem of expressing adenoviral genes that result in an immunological response to viral proteins when a gutless rAd vector is used in gene therapy.
  • Methods for producing gutless rAd vectors have been described, for example, in! U.S. Patent No. 5,981,225 to Kochanek et al., and U.S. Patent Nos.
  • ITRs inverted terminal repeats of adenovirus
  • the "inverted terminal repeats (ITRs) of adenovirus” are short elements located at the 5' and 3' termini of the linear adenoviral gqnome, respectively and are required for replication of the viral DNA.
  • the left ITR is located between 1- 130 bp in the Ad genome (also referred to as 0-0.5 mu).
  • the right ITR is located from about 3,7500 bp to the end of the genome (also referred to as 99.54100 mu).
  • the two ITRs are inverted repeats of each other.
  • the left ITR or 5' end is used to define the 5' and 3' ends of the ITRs.
  • the 5' end of the left ITR is located at the extreme 5' end of the linear adenoviral genome; picturing the left ITR as an arrow extending from the 5' end of the genome, the tail of the 5' ITR is located at mu 0 and the head of the left ITR is located at about 0.5 mu (further the tail of the left ITR is referred to as the 5' end of the left ITR and the head of the left ITRiis referred to as the 3' end of the left ITR).
  • the tail of the right or 3 1 ITR is located at rhu 100 and the head of the right ITR is located at about mu 99.5; the head of the right ITR is referred to as the 5' end of the right ITR and the tail of the right ITR is referred to as the 3' end of the right ITR.
  • the ITRs face each other with the head of each ITR pointing inward toward the bulk of the genome.
  • the tails of each ITR (which comprise the 5' end of the left ITR and the 3' end of the right ITR) are located in proximity to one another while the heads of each ITR are separated and face outward.
  • the "encapsidation signal of adenovirus” or “adenovirus packaging sequence” refers to the ⁇ sequence which comprises five (AI-AV): packaging signals and is required for encapsidation of the mature linear genome; theipackaging signals are located from about 194 to 358 bp in the Ad genome (about 0.5-1.0 m ⁇ ).
  • the viral backbone shuttle vector of the present invention contains a left and a right inverted terminal repeats of adenovirus, an encapsidation signal ( ⁇ ) of adenovirus, a pBR322 replication origin, a kanamycin resistance gene, and a stuffer sequence, which is the hypoxanthine phosphoribosyltransferase (HPRT) intron fragment ⁇ yith an approximately 10 kb. (SEQ ID NO: 1).
  • HPRT hypoxanthine phosphoribosyltransferase
  • the viral backbone shuttle vector of the present invention contains multiple restriction endonuclease sites for the insertion of a foreign DNA sequence of interest.
  • the viral backbone shuttle vector contains seven unique cloning sites where the foreign DNA sequence can be inserted by molecular cloning techniques that are well known in the DNA cloning art.
  • the foreign DNA sequence of interest typically comprises cDNA or genomic fragments that are of interest to transfer into mammalian cells.
  • Foreign DNA sequence of interest may include any naturally occurring or synthetic DNA sequence.
  • the foreign DNA may be identical in sequence to naturally-occurring DNA or may be mutated relative to'the naturally occurring sequence.
  • the foreign DNA need not be characterized asito sequence or function.
  • the size of foreign DNA that may be included in the shuttle vector will depend upon the size of the rest of the vector. If necessary, the st ⁇ ffer sequence may be removed to adapt large size foreign DNA fragment.
  • the total s'ize of foreign DNA may vary from lkb to 35kb.
  • the total size of foreign E)NA is from 15kb to 35 kb.
  • the foreign DNA may encode protein, or contain regulatory sites, including but not limited to, transcription factor binding sites, promoters, enhancers, silencers, ribosome binding sequences, recombination sites, origins of replication, sequences which regulate RNA stability and polyadenylation signals.
  • the promoters used may vary in their nature, origin and properties. The choice of promoter depends in fact on the desired use and on the gene of interest, in particular. Thus, the promoter may be constitutive or regulated, strong or weak, ubiquitous or tis.sue/cell-specific, or even specific of physiological or pathophysiological states (activity dependent on the state of cell differentiation or the step in the cell cycle).
  • the promoter may be of eukaryotic, prokaryotic, viral, animal, plant, artificial or human, etc., origin. Specific examples of promoters are the promoters of the genes PGK, TK, ⁇ 3H, ⁇ -EFl, APO, CMV, RSV etc. or artificial promoters, such as those for p53, E2F or cAMP.
  • the viral backbone shuttle vector of the present invention comprises at least 15 contiguous bases of SEQ IDNO: 1, preferably comprises at least 90 contiguous bases of SEQ ID NO: 1, more preferably comprises at least 300 contiguous bases of SEQ ID NO: 1 , and most preferably comprises 3000 or more contiguous bases of SEQ ID NO: 1.
  • One aspect of the present invention relates to a gutless adenoviral vector that carries a DNA sequence encoding a native TM protein or a variant of a TM protein.
  • the native TM protein is a human TM protein having the amino acid sequence recited in SEQ ID NO;2.
  • the DNA sequence is controlled by a regulatory element.
  • the regulatory element is a constitutive promoter such as the CMV promoter or RSV promoter.
  • the DNA sequence is controlled by a regulatable expression system. Systems to regulate expression of therapeutic, genes have been developed and incorporated into the current viral gene delivery vectors. These systems are briefly described below:
  • Tet-onloff system The Tet-system is based on two regulatory elements derived from the tetracycline-resistance operon of the E. coli Tn 10 transposon: the tet repressor protein (TetR) and the Tet operator DNA sequence (tet ⁇ ) to which TetR binds.
  • the system consists of two components, a “regulator” and, a “reporter” plasmid.
  • the "regulator” plasmid encodes a hybrid protein containing a mutated Tet repression (tetr) fused to the VP 16 activation domain of herpes simplex virus.
  • the "reporter” plasmid contains a tet-responsive element (TRE), which controls the "reporter” gene of choice.
  • TRE tet-responsive element
  • the tetr-VP16 fusion protein can onlyibind to the TRE, therefore activate the transcription of the "reporter” gene, in the presence of tetracycline.
  • the system has been incorporated into a number of viral vectors including retrovirus, adenovirus (Gossen and Bujard, PNAS ⁇ /&4189: 5547-5551 , [1992]; Gossen et al., Science 268: 1766-1769, [1995]; Kistner et al., PNAS USA 93: 10933-10938, [1996]). [073] Ecdysone system.
  • the Ecdysone system is based on the molting induction system found in Drosophila, but modified for inducible expression in mammalian cells.
  • the system uses an analog of the drosophila steroid hormone ecdysone, muristerone A, to activate expression of the gene of interest via a heterodimeric nuclear receptor. Expression levels have been reported to exceed 200- fold over basal levels with no effect on mammalian cell physiology (No et al., PNAS USA 93: 3346-3351 , [1996]).
  • Progesterone-system The progesterone receptor is normally stimulated to bind to a specific DNA sequence and to activate transcription through an interaction with its hormone ligand. Conversely, the progesterone antagonist mifepristone (RU486) is able to block hormone-induced nuclear transport and subsequent DNA binding. A mutant form of the progesterone receptor that can be stimulated to bind through an interaction with RU486 has been generated. To generate a specific, regulatable transcription factor, the RU486-binding domain of the progesterone receptor has been fused to the DNA-binding domain of the yeast transcription factor GAL4 and the transactivation domain of the KSV protein VPl 6. The chimeric factor is inactive in the absence of RU486.
  • Rapamycin-system Immunosuppressive agents, such as FK506 and rapamycin, act by binding to specific cellular proteins and facilitating their dimerization. For example, the binding of rapamycin to FK506-6inding protein (FKBP) results in its heterodimerization with another rapamycin .binding protein FRAP 1 which can be reversed by removal of the drug. The ability to bring two proteins together by addition of a drug potentiates the regulation of a number of biological processes, including transcription. A chimeric DNA-binding domain has been fused to the FKBP, which enables binding of the fusion protein to a specific DNA-binding sequence. A transcriptional activation domain also has been used to FRAP.
  • FKBP FK506-6inding protein
  • a fully functional transcription factor can be formed by heterodimerization mediated by addition of rapamycin.
  • the dimerized chimeric transcription factor can then bind to a synthetic promoter sequence containing copies of the synthetic DNA-binding sequence.
  • This system has been successfully integrated into adenoviral vectors. Long-term regulatable gene expression has been achieved in both' mice and baboons (Magari et al., J. Clin. Invest. 100: 2865-2872, [1997]; Ye et al., Science 283:88-91 , [1999]).
  • the instant invention uses a gutless adenovirus vector to express a native thrombomodulin protein or a variant of the thrombomodulin protein at a vessel graft or angioplasty site to prevent or reduce re-occlusion and intimal hyperplasia.
  • the amino acid sequence of human thrombomodulin (SEQ TD NO: 2) and the DNA sequence encoding human thrombomodulin (SEQ ID NO: 3) have been reported (Suzuki et al. EMBO J. 6: 1891-1897, [1987]).
  • the in vivo expression of thrombomodulin or a thrombomodulin variant is used for the treatment of atherosclerotic cardiovascular disease (CVD).
  • CVD atherosclerotic cardiovascular disease
  • venous grafts can be used for bypass surgeries, the veins eventually, become occluded by thrombosis resulting the recurrence of the diseases.
  • TM gene delivery is used in coronary artery bypass grafting, and vascular graft prostheses to block thrombosis.
  • TM gene is introduced into a segment of blood vessel in vitro using a gene transfer vector.
  • TM gene delivery can be also used for the reduction of no-intima formation, for the prevention of atherosclerosis; for the prevention of myocardial infarction and for the inhibition of fibrinolysis in hemophilic plasma.
  • TM gene transfer at the site of thrombus formation is potent approach to reverse these vascular diseases.
  • in vivo TM expression is achieved by embedding a gene transfer vector in a stent which is placed at the treatment site following percutaneous transluminal coronary angioplasty, peripheral artery angioplasty, thrombectomy, or an intravascular stenting procedure 1 .
  • the in vivo expression of thrombomodulin, or a thrombomodulin variant is used for the treatment of end stage renal failure (ESRD).
  • ESRD patients often exhibit decreased antithrombotic activity due 1 to low TM levels.
  • enhanced in vivo TM gene expression can be potentially very useful.
  • the in vivo TM expression Us achieved by administering a gene transfer vector to a mammal intravenously ( ⁇ v.), intramuscularly (i.m.), intraperitoneally (i.p.) or subcutaneously.
  • a gene transfer vector to a mammal intravenously ( ⁇ v.), intramuscularly (i.m.), intraperitoneally (i.p.) or subcutaneously.
  • intravenous administration often lead to viral infection of hepatocytes and transgene expression in the liver.
  • EXAMPLE 1 Construction of Gutless Viral Backbone Shuttle Vector pShuttle-ITR- HPRT
  • FIG. 1 An embodiment of a gutless viral backbone shuttle) vector pShuttle- ITR-HPRT is shown in Figure 1. Sequence portion containing R-JlTR, PBR322 ori, Kan, L-ITR, and encapsidation signal was obtained from the pAdEasy® system from STRATEGENE®. At bp 3667 of the original pShuttle sequence, there is a BamHI site just beyond the R-ITR. PCR primers were designed to include the BamHI site and then were to create an EcoRI site at the end of the R-ITR. The R-ITR was PCR replicated and then digested with BamHI and EcoRI to create sticky ends.
  • the viral backbone was then cut with both BamHI and EcoRI.
  • the BamHI icut the backbone at bp 3667 and there was also an EcoRI site inside the MCS at bp 377.
  • the backbone portion of the plasmid was then gel purified and the PCR replicated R-ITR was recloned into position. This essentially puts the L-ITR, encapsidation signal, MCS, and R-ITR all in close proximity to each other.
  • Insertion of the HPRT introns was a two step cloning process.
  • the viral backbone pShuttle-ITR was digested with EcoRI and Xbal, both enzyme sites are in the MCS.
  • the HPRT source was also digested with EcoRI and Xbal yielding a 7477 bp fragment that was cloned into the EcoRI/Xbal digested viral backbone.
  • the HPRT source was digested with only Xbal yielding a 2715 bp 1 fragment.
  • One of the Xbal sites in this cut is the same Xbal site that was cut from the EcoRI/Xbal double digest in step 1.
  • the viral backbone was cut with only Xbal and the 2715 bp fragment was inserted.
  • the HPRT stuffer sequence is inserted into the viral backbone in reverse orientation, hence intron 5, then 4, then 3.
  • the 2715 bp fragment was inserted and checked to follow the original source sequence.
  • the new plasmid is designated as pShuttle-ITR-HPRT (SEQ ID NO: 1)
  • EXAMPLE 2 Construction and Preparation of Gutless Viral Shuttle Vector Carrying Human Thrombomodulin or lacZ Gene
  • hTM The insertion of hTM into the gutless adenovirus backbone first required the creation of a CMV-hTM expression cassette.
  • the intermediate vector used was pcDNA3.1/Zeo(+) (Invitrogen).
  • a CMV promoter is available commercially and a CMV promoter was cloned into the multipleicloning sites (MCS) at the Xbal/EcoRV restriction enzyme site locations.
  • MCS multipleicloning sites
  • pcDNA3.1/Zeo(+) was cleaved insijde the MCS using both Xbal and EcoRV as well.
  • the CMV promoter was then ligated.
  • the CMV promoter (SEQ ID NO:4) was inserted in a backwards orientation relative to the pcDNA3.1/Zeo (+) plasmid.
  • the human TM cDNA (SEQ ID NO:5) was obtained from Dr. Sadlerl(Dittman et al., Biochemistry, 26(14):4350-4357 [1987]) which the sequence was also submitted to ATCC and to GenBank.
  • the human TM gene was removed from' the plasmid using EcoRI and inserted into pcDNA3.1/Zeo(+), also in the reverse orientation to pcDNA3.1/Zeo(+) downstream of the inserted CMV promoter.
  • the expression cassette in pCMV-hTM was removed by digesting with Pmel.
  • the gutless adenovirus backbone pshuttle-ITR-HPRT was; linearized using Smal which cuts the plasmid at bp 381.
  • the CMV-hTM cassette was ligated to the gutless virus in the forwards orientation. Sequence of the expression cassette (from Pmel site to Pmel site) is shown in SEQ ID NO:6, The new plasmid is designated as pShuttle-ITR-HPRT-CMV-TM.
  • Amplification of a human DNA sample resulted in. the amplification of a 18524bp DNA fragment (stuffer 1, SEQ ID NO: 12).
  • Stuffer 1 was cut with the restriction enzymes BstEII and Sfil and the resulting fragment of approximately 18371 bp was inserted into the BsteII and Sfil sites of pTMadap, resulting in pTMadap- stufferl.
  • pTMadap-stufferl was digested with SanDI and BstEII and the resulting DNA ends were modified by a fill-in reaction with Klenow. Re-ligation resulted in the 25207 bp vector pTMadap-stufferl -short.
  • the sequence of stufferl -short fragment is shown in SEQ ID NO: 13.
  • the plasmid p2-2 (SEQ ID NO: 14, obtained frormGenBank) was cut with Notl and the resulting fragment of approximately 5954 bp (stuffer 2, SEQ ID NO: 15) was inserted into the Notl site of pTMadap-stufferl short', resulting in pTMadap-stufferl -short-stuffer2.
  • Plasmid pTMadap-stufferl -short-stuffer2 was cut with AcII and BsiWl .
  • the resulting 28790 bp fragment was isolated from gel.
  • pShuttle-ITR-HPRT (SEQ ID NO:1) was cut with AcIl and Acc65I.
  • the resulting 1966 bp fragment was ligated into the isolated 28790 bp fragment, resulting in the full length backbone vector pTM-final ( Figure 2 and SEQ ID NO: 16).
  • LacZ also required creation of an intermediate vector to create the expression cassette.
  • pcDNA3.1/Zeo (+) was again used.
  • the LacZ gene was inserted into the vector MCS using Notl/Xbal.
  • EXAMPLE 3 Preparation of gutless adenovirus carrying humani thrombomodulin gene (gutless Ad.hTM " )
  • the gutless Ad.hTM was prepared according to th'e following protocol: [096] 1. Linearize pTM-final by digestion with Pad. The completeness of the digestion is confirmed by electrophoresis using a small aliquot of the digestion product. It's not necessary to gel purify the digested pTM-final for transfection described in step 2).
  • 293FLP cells grown in a 60mm disWat about 80% confluence with about 5 ⁇ g of Pad-digested pTM-final using lip ⁇ fectamine.
  • 293FLP cells are 293 cells engineered to express the flp gene product, which recognizes the FRS flanking the encapsidation signal and cleaves out the encapsidation signal thereby not allowing helper-viral DNA to be packaged.
  • helpervirus HlO in 2% DMEM-F12 at a multiplicity of infection (MOI) of 10.
  • [0106] 1 Transfer the supernatant, which contains the released virus, to a fresh sterile culture tube and subject the supernatant to a second'round of centrifugation to further remove cell debris.
  • CsCI was prepared to density 1.33 g/ml.
  • Two fresh ultra-clear tubes were placed 8 mL of CsCl and overlay the band just recovered after the first spin. (To equilibrate the tubes, measure, before the volume of the recovered band and divide equally in the 2 tubes). Samples iwere centrifuged at the conditions above for 18 hours. The opalescent band was recovered and collected in a sterile eppendorf tube. (From this moment, keep the tube always on ice).
  • BioRad protein estimation kit was used with 1:5 diluting, and placing 1 ml in each disposable cuvette. Standards were set up at 0, 1, 2, 5 10, and 15 ⁇ g/ml. (BSA is fine). Sample cuvettes were prepared using 1-10 ⁇ l of sample, depending on estimate of titer. (Sample OD must be within the linear range of the standard line,) OD was taken at 595 ⁇ and formula of the line was calculated from 1 standards. The protein concentration of the samples was calculated using this for ⁇ ula. The following formula was used to convert protein concentration to titer: [12.956 + 224.15 ( ⁇ g/ml)] X lO 8 .
  • EXAMPLE 4 Expression of human thrombomodulin ChTM) in vitro (A) Expression of hTM in HEK 293 cells transfected with pTM-final
  • HEK 293 cells were cultured in a 6 well cluster and transfected with 1 ⁇ g of pTM-final. After 24 hours, the cells were washed with PBS and lysed in 125 ⁇ l RlPA buffer with protease inbitors Protein samples (16 ⁇ l) were separated on 7.5% polyacrylamide/ SDS gel and transferred to nitrocellulose membrane.
  • Primary antibody TM (c-17) (1 :2000, Santa Cruz) and secondary antibody Polyclonal Rabbit Anti-Goat Immunoglobulins/HRP (1 :4000, DakoCytomation) waS'Used to detect the proteins. As shown in Figure 3, hTM expression was detectable iri cells transfected with pTM-final.
  • the RIPA buffer was prepared according the following recipe: mixing 100 ⁇ l Igepal ca-630, 50 mg sodium deoxycholate, 500 ⁇ l 20 % SDS, 10 mM ⁇ - mercapto ethanol, and 1 ml 10x PBS, and add water to a final volume of 10 ml at room temperature.
  • the P2 lysate was generated as described in Example 3. After CPE was observed, 293FLP cells were detached from the bottom of trie culture flask by repeated tapping of the flask. 1 m! of the total of 10 ml of cell suspension was used for the detection of TM expression. The cells in the ImI cell suspension were collected by centrifugation for 10 min at 300xg and lysed in 250' ⁇ l RIPA buffer. 7ul of 5x loading buffer was added to 35 ⁇ l of the lysed cells and the; resulting solution was immersed in boiling water for 3 minutes.
  • the 5x loading buffer was prepared by mixing 20.0 ml 30% SDS, 11.5 ml 2M sucrose, 6.5 ml 2M Tris-HCL pH 6.8, 2.0 ml beta-mercaptoethanol and bromophenolblue.
  • the RlPA buffer was prepared as described in Example 4(A).
  • C Expression of TM in virus infected vena cava
  • Vena cava was excised from rats and cut into six segments of approximately 3mm long. The segments were incubated for 30 m'inutes in medium containing gutless luc or TM virus. After incubation, the segments were washed three times and transferred to a 24-well plate containing DMEM. The segments were incubated overnight in an atmosphere of 95%C> 2 and 5%CO 2 with gentle shaking. After 24 hours of incubation the segments were frozen. The frozen sections were thawed in lysis buffer and loaded onto a 7.5% SDS acrylamide gel. After blotting, the blot was probed with an antibody against human TM.
  • HUVEC cells will be infected the gutless adenovirus expressing LacZ. These cells will subsequently be stained with X-gal to look for blue cells, This will demonstrate the viability of the gutless adenovirus backbone itself.
  • HEK 293 cells were cultured in a 6 well cluster ancl transfected with 200 ⁇ l of TM gutless virus of passage 1-6. After 24 hours, the cells were washed with PBS and lysed in 125 ⁇ l RIPA buffer. Protein samples (16 ⁇ l) were separated on a 7.5% polyacrylamide/ SDS gel and transferred to nitrocellulose membrane. Primary antibody TM (c-17) (1 :2000, Santa Cruz) and secondary antibody, Polyclonal Rabbit Anti-Goat Jmmunoglobulins/HRP (1 :4000, DakoCytomation) were used to detect the proteins. As shown in Figure 6, TM expression is higher in cells infected with virus of higher passage numbers , indicating successful amplification of TM gutless virus in 293CRE cells.
  • the RIPA buffer (10 ml) was prepared as follows: 100 ⁇ l Igepal ca- 630, 50 mg sodium deoxycholate, 500 ⁇ l 20 % SDS, 10 mM ⁇ -mercapto ethanol, 1 ml 10x PBS, add water to make up 10 ml. Immediately before use, the following protease inhibitors were added to the RIPA buffer: 115 ⁇ l PMSFi(from 34,8 mg/ml in
  • the Complete Viral Delivery System composes of 1 : 1 mixture of Ham's F12 medium and DMEM, an effective amount of a gutless virus vector carrying a polynucleotide encoding a thrombomodulin protein or a variant of a thrombomodulin protein, and an a cellular oxygen carrier.
  • Preferred oxygen carrier includes: unmodified or chemically modified hemoglobin in the range of 3 g/dl to 10 g/dl and perfluorochemical emulsions.
  • the CVDS may optionally contain 1 mM L- glutamine (Sigma), 1.5 g/L sodium bicarbonate (Sigma), IX antibiotic-antimycotic (GEBCO® 15240). The CVDM maintains tissue viability during the viral treatment of blood vessel.
  • a vein segment is harvested from the leg and is stored in Ham's F 12 medium.
  • Gutless adenovirus suspended in CVDM is then injected into the isolated vein segment and incubated for 10 to 40 minutes depending on the desired level of transfection. The infection may be performed under pressure to enhance efficiency.
  • the vein segment is washed several times to eliminate all viral particles that have not entered the endothelial .cells of the vein segment, and is then grafted into the desired treatment site. The' thorough rinse avoids the spread of the viral vector to other organs of the body following in situ grafting, and any systemic immune response to the viral vector.
  • the vein in the leg is treated following evacuation of the clot.
  • a catheter is inserted in the leg vein and both the proximal and distal balloons are inflated to isolate the vein segment to be transfected.
  • the segment is evacuated of all blood, rinsed with physiologic saline.
  • the segment is then filled with the CVDS described above, under pressure.
  • the isolated vein segment is exposed to the CVDS for a period of 10 to 45 minutes, depending upon the desired transfection efficiency.
  • the vein in the kidney is treated following evacuation of the clot.
  • a catheter is inserted in the kidney vein and both the proximal and distal balloons are inflated to isolate the vein segment to be transfected.
  • the segment is evacuated of all blood, rinsed with physiologic saline; it is then filled with the CVDS described above, under pressure.
  • the isolated veih segment is exposed to the CVDS for a period of 10 to 45 minutes, depending upon the desired transfection efficiency.
  • a virus-coated stent is pla'ced at a treatment site after angioplasty.
  • the virus is a gutless adenovirus carrying a polynucleotide encoding a thrombomodulin protein or a variant of a thrombomodulin iprotein.
  • the virus may be embedded in the stent and is releases gradually through a time-releasing mechanism well-known to one skilled in the art.
  • EXAMPLE 10 In vivo expression of TM by local infusion of viral vectors.
  • the tail vein of experimental rats was flushed with a solution containing a gutless adenoviral vector carrying a luciferase transgene. As shown in Figure 7, the expression of luciferase was still very strong in the tail vein eight days after viral infection.
  • gutless TM virus 3 male wistar rats weighing approximately 300 grams were intravenously injected in the tail vein with a low dose of gutless TM virus (approximately 2e] 0 viral particles) in a total volume of 500 ul of sucrose buffer, After three weeks, the animals were sacrificed and liver tissue and blood plasma was collected and immediately frozen in liquid nitrogen.
  • Tm expression in the liver was determined by western blotting. Approximately 500mg of liver tissue was homogenized in 2 ml of RIPA buffer. Liver protein samples (20 ⁇ g) were separated on a 7.5% polyacrylamide/ SDS gel and transferred to nitrocellulose membrane. Primary antibody TM (c-17) (1 :2000, Santa Cruz) and secondary antibody Polyclonal Rabbit Anti-Goat Immunoglobulins/HRP (1 :4000, DakoCytomation) were used to detect the proteins.
  • HEK 293 cells were cultured in a 6 well cluster. 3 wells were infected with 100 ⁇ l of TM gutless virus (approximately 4e9 Vp) and 3 wells received no virus. After 24 hours, non-infected and TM infected cells were washed with PBS and lysed in 125 ⁇ l RIPA buffer. Protein samples (16 ⁇ l) were separated on a 7i5% polyacrylamide/ SDS gel and transferred to nitrocellulose membrane.
  • Blots containing protein from both TM expressing cells and non-infected cells were incubated with primary antibody TM (c-17) (1 :2000, Santa Cruz) or plasma from TM infected rats (1 :20, 1 : 100 and 1 : 1000 dillution). Detection of primary antibodies was performed using Polyclonal Rabbit Anti-Goat Immunoglobulins/HRP (1 :4000, DakoCytomation) and Polyclonal Rabbit Anti-Rat Immunoglobulins/HRP (1 :4000, DakoCytomation), respectively.
  • RIPA buffer was prepared as described in Example 4.
  • TM expression in the liver No adverse effects of'the injection of gutless TM virus could be detected. Animals displayed normal growth characteristics and did not suffer from excessive bleeding. Three weeks after injection, animals were , sacrificed and no internal bleeding could be detected. Liver TMjexpression was evaluated using western-blot. TM expression was elevated two-fold above background levels, indicating modest over-expression of TM giitless virus in the liver three weeks after infection (Figure 8).

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Abstract

L'invention concerne des procédés et des compositions destinés au traitement de maladies cardiovasculaires et vasculaires périphériques faisant appel à des technologies d'administration génique ex vivo et in vivo. Dans un aspect, l'invention concerne un procédé permettant de traiter une maladie vasculaire par introduction d'une séquence ADN codant une protéine TM ou son variant dans un segment d'un vaisseau sanguin in vivo au moyen d'un vecteur adénoviral n'exprimant plus de gènes viraux. Dans un autre aspect, l'invention concerne un procédé permettant d'administrer un vecteur adénoviral n'exprimant plus de gènes viraux transportant une séquence ADN codant une protéine TM ou son variant au moyen d'une endoprothèse.
PCT/US2007/006371 2007-01-08 2007-03-14 Expression ex vivo et in vivo du gène thrombomoduline pour le traitement de maladies cardiovasculaires et vasculaires périphériques WO2008088358A2 (fr)

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EP07772782A EP2099438A4 (fr) 2007-01-08 2007-03-14 Expression ex vivo et in vivo du gène thrombomoduline pour le traitement de maladies cardiovasculaires et vasculaires périphériques
CA2674563A CA2674563C (fr) 2007-01-08 2007-03-14 Expression ex vivo et in vivo du gene thrombomoduline pour le traitement de maladies cardiovasculaires et vasculaires peripheriques

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US11/650,478 US7501114B2 (en) 2002-12-02 2007-01-08 Ex vivo and in vivo expression of the thrombomodulin gene for the treatment of cardiovascular and peripheral vascular diseases
US11/650,478 2007-01-08
US11/650,479 US7481998B2 (en) 2002-12-02 2007-01-08 Ex vivo and in vivo expression of the thrombomodulin gene for the treatment of cardiovascular and peripheral vascular diseases
US11/650,479 2007-01-08
US11/685,474 2007-03-13
US11/685,474 US7803365B2 (en) 2002-12-02 2007-03-13 Ex vivo and in vivo expression of the thrombomodulin gene for the treatment of cardiovascular and peripheral vascular diseases

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WO2008088358A3 WO2008088358A3 (fr) 2008-12-18

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US7132277B1 (en) * 2000-01-31 2006-11-07 Merck & Co., Inc. Helper dependent vector system for gene therapy
GB2410747A (en) * 2002-12-02 2005-08-10 Biovec Llc Ex vivo and in vivo expression of the thrombomodulin gene for the treatment of cardiovascular and peripheral vascular diseases
US20050106124A1 (en) * 2003-02-25 2005-05-19 Sehgal Lakshman R. Therapeutic applications of thrombomodulin gene via viral and non-viral vectors

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Also Published As

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CA2674563A1 (fr) 2008-07-24
EP2099438A2 (fr) 2009-09-16
WO2008088358A3 (fr) 2008-12-18
CA2674563C (fr) 2014-12-30
EP2099438A4 (fr) 2011-09-28

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