EP1115733A1 - Sequences caracteristiques de la transcription genique regulee par l'hypoxemie - Google Patents

Sequences caracteristiques de la transcription genique regulee par l'hypoxemie

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Publication number
EP1115733A1
EP1115733A1 EP99945523A EP99945523A EP1115733A1 EP 1115733 A1 EP1115733 A1 EP 1115733A1 EP 99945523 A EP99945523 A EP 99945523A EP 99945523 A EP99945523 A EP 99945523A EP 1115733 A1 EP1115733 A1 EP 1115733A1
Authority
EP
European Patent Office
Prior art keywords
hypoxia
polynucleotide
gene
functional analogs
seq
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Withdrawn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
EP99945523A
Other languages
German (de)
English (en)
Other versions
EP1115733A4 (fr
Inventor
Paz Einat
Rami Skaliter
Elena Feinstein
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.)
Quark Pharmaceuticals Inc
Original Assignee
Kohn Kennedy I
Quark Biotech 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 Kohn Kennedy I, Quark Biotech Inc filed Critical Kohn Kennedy I
Publication of EP1115733A1 publication Critical patent/EP1115733A1/fr
Publication of EP1115733A4 publication Critical patent/EP1115733A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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

Definitions

  • the present invention relates to the identification of genes that are differentially expressed in hypoxia and use of the genes and gene products for diagnosis and therapeutic intervention.
  • the invention further relates to identification of polynucleotide sequences that are differentially expressed in hypoxia and the use of the sequences for diagnosis and probes.
  • tissue oxygenation plays an important role in normal development as well as in pathologic processes such as ischemia. Tissue oxygenation plays a significant regulatory/inducer role in both apoptosis and in angiogenesis (Bouck et al, 1996; Bunn et al, 1996; Dor et al, 1997; Carmeliet et al, 1998) . Apoptosis (see Duke et al, 1996 for review) and growth arrest occur when cell growth and viability are reduced due to oxygen deprivation (hypoxia). Angiogenesis (i.e. blood vessel growth, vascularization) is stimulated when hypo-oxygenated cells secrete factors which stimulate proliferation and migration of endothelial cells in an attempt to restore oxygen homeostasis (for review see Hanahan et al, 1996) .
  • Angiogenesis i.e. blood vessel growth, vascularization
  • hypoxia plays a critical role in the selection of mutations that contribute to more severe tumorogenic phenotypes (Graeber et al . , 1996). Identifying activated or inactivated genes and gene products in hypoxia and ischemia is needed. Ischemic disease pathologies involve a decrease in the blood supply to a bodily organ, tissue or body part generally caused by constriction or obstruction of the blood vessels, as for example retinopathy, myocardial infarction and stroke. Therefore, apoptosis and/or angiogenesis as induced by the ischemic condition are also involved in these disease states. Neoangiogenesis is seen in some forms of retinopathy and in tumor growth. These processes are complex cascades of events controlled by many different genes reacting to the various stresses such as hypoxia.
  • hypoxia-triggered activation of genes can provide a tool to identify not immediately evident ischemia in a patient. Identification of hypoxia-regulated genes permits the utilization of gene therapy or direct use of gene products, or alternatively inactivation of target genes for therapeutic intervention in treating the diseases and pathologies associated with hypoxia, ischemia and tumor growth.
  • the present invention provides purified, isolated and cloned polynucleotides (nucleic acid sequences) associated with hypoxia-regulated activity and having sequences designated as any one of SEQ ID NOS. 1-9, or having complementary or allelic variation sequences thereto.
  • the present invention provides a method of regulating angiogenesis or apoptosis in a patient in need of such treatment by administering to such patient a therapeutically effective amount of an antagonist of at least one protein as encoded by the nucleic acid sequences as set forth in any of SEQ ID NOS. 1-9.
  • the diagnostic tool for identifying genes modulated by hypoxic conditions having a detector for detecting the presence of a polynucleotide having a nucleic acid sequence according to any of SEQ. ID NOS. 1-9.
  • a pharmaceutical composition for modulating hypoxia and ischemia having an effective amount of a polynucleotide having the nucleic acid sequence according to SEQ. ID NOS. 1-9 and a pharmaceutically acceptable carrier is also provided.
  • hypoxia response regulating genes there are provided hypoxia response regulating genes.
  • Figure 1 shows the nucleic acid sequence of the 92 gene (Seq. I.D. No. 1) ;
  • Figure 2 shows the nucleic acid sequence of the 95 gene (Seq. I.D. No. 2)
  • Figure 3 shows the nucleic acid sequence of the 98 gene (Seq. I.D. No. 3) ;
  • Figure 4 shows the nucleic acid sequence of the 60F6 gene (Seq. I.D. No. 4);
  • FIGS 5 A-C show the nucleic acid sequence of the
  • Figure 6 shows the nucleic acid sequence of the 24D4 gene (Seq. I.D. No. 6);
  • Figures 7 A and B show the nucleic acid sequence of the 77H4 gene (Seq. I.D. No. 7) ;
  • Figure 8 shows the nucleic acid sequence of the 14G2 gene (Seq. I.D. No. 8).
  • Figure 9 shows the nucleic acid sequence of the 29F3 gene (Seq. I.D. No. 9).
  • nucleic acid sequences with sequences as set forth herein in SEQ. ID Nos . 1-9, that can be utilized diagnostically in hypoxia and ischemia and that can be used as targets for therapeutic intervention, or can be used to identify genes that are regulated and respond to hypoxic conditions.
  • SEQ ID Nos. 1-4 and 6-8 have not previously been identified.
  • SEQ ID No. 5 was found to match sequences in data banks but has not been reported to be associated with hypoxia regulation.
  • the present invention further provides candidate genes and gene products that can be utilized therapeutically and diagnostically in hypoxia and ischemia and that can regulate apoptosis or angiogenesis.
  • regulate or modulate or control it is meant that the process is either induced or inhibited to the degree necessary to effect a change in the process and the associated disease state in the patient . Whether induction or inhibition is being contemplated will be apparent from the process and disease being treated and will be known to those skilled in the medical arts.
  • the present invention identifies genes for gene therapy, diagnostics and therapeutics that have direct causal relationships between a disease and its related pathologies and up- or down-regulator (responder) genes. That is, the present invention is initiated by a physiological relationship between cause and effect.
  • the present invention also provides a method of regulating angiogenesis or apoptosis in a patient in need of such treatment by administering to such patient a therapeutically effective amount of an antagonist of at least one protein as encoded by the nucleic acid sequences as set forth in any of SEQ ID NOS. 1-9.
  • sequences are partial gene sequences which are markers/probes for genes that are upregulated under hypoxic conditions. These partial sequences can be designated "Expressed Sequence Tags"
  • ESTs are markers for the genes actually expressed in vivo and are ascertained as described herein in the Examples or as is known in the art.
  • ESTs comprise DNA sequences corresponding to a portion of nuclear encoded mRNA.
  • the EST has a length that allows for PCR (polymerase chain reaction) , for use as a hybridization probe and is a unique designation for the gene with which it hybridizes (generally under conditions sufficiently stringent to require at least 95% base pairing) .
  • PCR polymerase chain reaction
  • ESTs and their functional utility see, WO 93/00353 PCT Application which is incorporated herein in its entirety by reference, as well as the references by Zweiger et al, 1997; Okubo et al, 1997 and Braren et al, 1997.
  • the WO 93/00353 PCT application further describes how the EST sequences can be used to identify the transcribed genes.
  • the present invention also provides a method of diagnosing the presence of ischemia or other hypoxia- associated pathologies in a patient including the steps of analyzing a tissue sample from the patient for the presence of at least one expressed gene (up-regulated) identified by the sequences of the present invention utilized as probes.
  • Methods of identification of hybridization can include immunohistochemical staining of the tissue samples.
  • Southern blotting, single strand conformational polymorphism, restriction endonuclease fingerprinting (REF) , PCR amplification and DNA-chip analysis using the nucleic acid sequences of the present invention as probes/primers can be used.
  • hypoxia-responding genes identified by the probes/sequences hybridizing under stringent conditions with 95% homology set forth herein or a complementary or allelic variation sequence and human homologies as needed thereto are disclosed.
  • the present invention further provides proteins as encoded by the identified genes.
  • the present invention further provides antibodies directed against these proteins.
  • the present invention further provides transgenic animals and cell lines carrying at least one expressible gene identified by the present invention.
  • the present invention further provides knock-out eucaryotic organisms in which at least one nucleic acid sequences as identified by the probes of the present invention.
  • the present invention provides a method of regulating angiogenesis, apoptosis or other hypoxia- associated pathologies in a patient in need of such treatment by administering to a patient a therapeutically effective amount of an antagonist of at least one protein as encoded by the nucleic acid sequences or sequences identified herein or by the probes of the present invention, or alternatively by a non-protein product of the gene's activity, or inactivation of a gene by chemical compound.
  • the present invention provides a method of regulating angiogenesis, apoptosis or other hypoxia-associated pathologies in a patient in need of such treatment by administering to a patient a therapeutically effective amount of at least one antisense oligonucleotide against the nucleic acid sequences or dominant negative peptide directed against the sequences or their proteins .
  • the present invention further provides a method of regulating angiogenesis or apoptosis in a patient in need of such treatment by administering to a patient a therapeutically effective amount of a protein encoded by the identified genes as active ingredients in a pharmaceutically acceptable carrier.
  • the present invention provides a method of providing an apoptosis-regulating gene, an angiogenesis- regulating gene or hypoxia-response regulating gene identified by the probes of the present invention, by administering directly to a patient in need of such therapy an expressible vector comprising expression control sequences operably linked to one of the identified genes and its human homolog if appropriate .
  • the proteins of the present invention can be produced recombinantly (see generally Marshak et al, 1996 "Strategies for Protein Purification and Characterization. A laboratory course manual.” CSHL Press) and analogues can be due to post-translational processing.
  • Analogue as used herein is defined as a nucleic acid sequence or protein which has some differences in their amino acid/nucleotide sequences as compared to the native sequence of SEQ. ID NOS. 1-9. Ordinarily, the analogue will be generally at least 70% homologous over any portion that is functionally relevant . In more preferred embodiments the homology will be at least 80% and can approach 95% homology to the protein/nucleotide sequence.
  • amino acid or nucleotide sequence of an analogue can differ from that of the primary sequence when at least one residue is deleted, inserted or substituted, but the protein or nucleic acid molecule remains functional. Differences in glycosylation can provide protein analogues .
  • Functionally relevant refers to the biological property of the molecule and in this context means an in vivo effector or antigenic function or activity that is directly or indirectly performed by a naturally occurring protein or nucleic acid molecule.
  • Effector functions include but are not limited to include receptor binding, any enzymatic activity or enzyme modulatory activity, any carrier binding activity, any hormonal activity, any activity in promoting or inhibiting adhesion of cells to extracellular matrix or cell surface molecules, or any structural role as well as having the nucleic acid sequence encode functional protein and be expressible.
  • the antigenic functions essentially mean the possession of an epitope or antigenic site that is capable of cross- reacting with antibodies raised against a naturally occurring protein.
  • Biologically active analogues share an effector function of the native which can, but need not, in addition possess an antigenic function.
  • the antibodies can be either monoclonal, polyclonal or recombinant and be used in immunoassays .
  • the antibodies can be prepared against the immunogen or portion thereof for example a synthetic peptide based on the sequence, or prepared recombinantly by cloning techniques or the natural gene product and/or portions thereof can be isolated and used as the immunogen.
  • Immunogens can be used to produce antibodies by standard antibody production technology well known to those skilled in the art as described generally in Harlow and Lane, Antibodies : A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988 and Borrebaeck, Antibody Engineering - A Practical Guide, W.H. Freeman and Co., 1992.
  • Antibody fragments can also be prepared from the antibodies and include Fab, F(ab') 2 , and Fv by methods known to those skilled in the art .
  • polyclonal antibodies For producing polyclonal antibodies a host, such as a rabbit or goat, is immunized with the immunogen or immunogen fragment, generally with an adjuvant and, if necessary, coupled to a carrier; antibodies to the immunogen are collected from the sera. Further, the polyclonal antibody can be absorbed such that it is monospecific . That is, the sera can be absorbed against related immunogens so that no cross-reactive antibodies remain in the sera rendering it monospecific.
  • the technique involves hyperimmunization of an appropriate donor with the immunogen, generally a mouse, and isolation of splenic antibody producing cells. These cells are fused to a cell having immortality, such as a myloma cell, to provide a fused cell hybrid which has immortality and secretes the required antibody. The cells are then cultured, in bulk, and the monoclonal antibodies harvested from the culture media for use .
  • RNAs from antibody producing B-lymphocytes of animals, or hybridoma are reverse-transcribed to obtain complimentary DNAs (cDNAs) .
  • Antibody cDNA which can be full or partial length, is amplified and cloned into a phage or a plasmid.
  • the cDNA can be a partial length of heavy and light chain cDNA, separated or connected by a linker.
  • the antibody, or antibody fragment is expressed using a suitable expression system to obtain recombinant antibody.
  • Antibody cDNA can also be obtained by screening pertinent expression libraries.
  • the antibody can be bound to a solid support substrate or conjugated with a detectable moiety or be both bound and conjugated, as is well known in the art.
  • the detectable moieties contemplated with the present invention can include, but are not limited to, fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, ⁇ - galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, 14 C and iodination.
  • fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, ⁇ - galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, 14 C and iodination.
  • transgenics and knock-outs of the present invention are constructed using standard methods knowr. in the art and as set forth in United St tes Patents 5,487,992, 5,464,764, 5,387,742, 5,360,735, 5,347,075, 5,298,422, 5,288,846, 5,221,778, 5,175,385, 5,175,384, 5,175,383, 4,736,866 as well as Burke and Olson (1991), Capecchi (1989), Davies et al . (1992), Dickinson e al . (1993), Duff and Lincoln (1995), Huxley et al . (1991), Jakobovits et al . (1993), Lamb et al .
  • any techniques known in the art can be used to introduce the transgene expressibly into animals to produce the parental lines of animals.
  • Such techniques include, but. are not limited to, pronuclear microinjection (U.S. patent 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al . , 1985); gene, targeting in embryonic stem cells (Thompson et al . , 1989; Mansour, 1990 and U.S. patent 5,614,396); electroporation of embryos (Lo, 1983); and sperm-mediated gene transfer (Lavitrano et al . , 1989).
  • pronuclear microinjection U.S. patent 4,873,191
  • retrovirus mediated gene transfer into germ lines Van der Putten et al . , 1985
  • gene, targeting in embryonic stem cells Thompson et al . , 1989; Mansour, 1990 and U.S. patent 5,614,396
  • one parent strain instead of carrying a direct human transgene can have the homologous endogenous gene modified by gene targeting such that it approximates the transgene. That is, the endogenous gene has been "humanized” and/or mutated (Reaume et al, 1996) . It should be noted that if the animal and human sequence are essentially homologous a "humanized” gene is not required.
  • the transgenic parent can also carry an over expressed sequence, either the non-mutant or a mutant sequence and humanized or not as required. The term transgene is therefore used to refer to all these possibilities.
  • cells can be isolated from the offspring which carry a transgene from each transgenic parent and that are used to establish primary cell cultures or cell lines as is known in the art.
  • a parent strain will be homozygous for the transgene.
  • the endogenous non-transgene in the genome that is homologous to the transgene will be non- expressive.
  • non-expressive is meant that the endogenous gene will not be expressed and that this non- expression is heritable in the offspring.
  • the endogenous homologous gene could be "knocked-out" by methods known in the art.
  • the parental strain that receives one of the transgenes could carry a mutation at the endogenous homologous gene rendering it non-expressed.
  • the antagonist/regulating agent/active ingredient is dosed and delivered in a pharmaceutically acceptable carrier as described herein below.
  • the term antagonist or antagonizing is used in its broadest sense.
  • Antagonism can include any mechanism or treatment which results in inhibition, inactivation, blocking or reduction in gene activity or gene product. It should be noted that the inhibition of a gene or gene product can provide for an increase in a corresponding function that the gene or gene product was regulating.
  • the antagonizing step can include blocking cellular receptors for the gene products and can include antisense treatment as discussed herein below.
  • a patient can be in need of inducing apoptosis in tumorogenic cells or angiogenesis in trauma situations where for example a limb must be reattached or in a transplant where revascularization is needed.
  • AS antisense
  • Many reviews have covered the main aspects of antisense (AS) technology and its enormous therapeutic potential (Wright and Anazodo, 1995) .
  • AS oligonucleotide sequences can be short sequences of DNA, typically 15-30 mer but can be as small as 7 mer (Wagner et al, 1996) , designed to complement a target mRNA of interest and form an RNA:AS duplex. This duplex formation can prevent processing, splicing, transport or translation of the relevant mRNA.
  • certain AS nucleotide sequences can elicit cellular RNase H activity when hybridized with their target mRNA, resulting in mRNA degradation (Calabretta et al , 1996) . In that case, RNase H will cleave the RNA component of the duplex and can potentially release the AS to further hybridize with additional molecules of the target RNA.
  • sequence target segment for the antisense oligonucleotide is selected such that the sequence exhibits suitable energy related characteristics important for oligonucleotide duplex formation with their complementary templates, and shows a low potential for self-dimerization or self-complementation [Anazodo et al., 1996] .
  • the computer program OLIGO (Primer Analysis Software, Version 3.4), can be used to determine antisense sequence melting temperature, free energy properties, and to estimate potential self-dimer formation and self-complimentary properties.
  • the program allows the determination of a qualitative estimation of these two parameters (potential self-dimer formation and self- complimentary) and provides an indication of "no potential” or "some potential” or “essentially complete potential” .
  • target segments are generally selected that have estimates of no potential in these parameters.
  • segments can be used that have "some potential” in one of the categories.
  • a balance of the parameters is used in the selection as is known in the art.
  • the oligonucleotides are also selected as needed so that analogue substitution do not substantially affect function.
  • Phosphorothioate antisense oligonucleotides do not normally show significant toxicity at concentrations that are effective and exhibit sufficient pharmacodynamic half-lives in animals (Agarwal et al . , 1996) and are nuclease resistant. Antisense induced loss-of-function phenotypes related with cellular development were shown for the glial fibrillary acidic protein (GFAP) , for the establishment of tectal plate formation in chick (Galileo et al . , 1991) and for the N-myc protein, responsible for the maintenance of cellular heterogeneity in neuroectodermal cultures (epithelial vs.
  • GFAP glial fibrillary acidic protein
  • Antisense oligonucleotide inhibition of basic fibroblast growth factor (bFgF) having mitogenic and angiogenic properties, suppressed 80% of growth in glioma cells (Morrison, 1991) in a saturable and specific manner. Being hydrophobic, antisense oligonucleotides interact well with phospholipid membranes (Akhter et al . , 1991). Following their interaction with the cellular plasma membrane, they are actively (or passively) transported into living cells (Loke et al . , 1989) , in a saturable mechanism predicted to involve specific receptors (Yakubov et al . , 1989) .
  • bFgF basic fibroblast growth factor
  • Ribozymes can be utilized. This is particularly necessary in cases where antisense therapy is limited by stoichiometric considerations (Sarver et al . , 1990, Gene Regulation and Aids, pp. 305-325) . Ribozymes can then be used that will target the same sequence . Ribozymes are RNA molecules that possess RNA catalytic ability (see Cech for review) that cleave a specific site in a target RNA. The number of RNA molecules that are cleaved by a ribozyme is greater than the number predicted by stochiochemistry. (Hampel and Tritz, 1989; Uhlenbeck, 1987) .
  • Ribozymes catalyze the phosphodiester bond cleavage of RNA.
  • ribozyme structural families include Group I introns, RNase P, the hepatitis delta virus ribozyme, hammerhead ribozymes and the hairpin ribozyme originally derived from the negative strand of the tobacco ringspot virus satellite RNA (sTRSV) (Sullivan, 1994; U.S. Patent No. 5,225,347, columns 4-5) .
  • the latter two families are derived from viroids and virusoids, in which the ribozyme is believed to separate monomers from oligomers created during rolling circle replication (Symons, 1989 and 1992) .
  • ribozyme motifs are most commonly adapted for trans-cleavage of mRNAs for gene therapy (Sullivan, 1994) .
  • the ribozyme type utilized in the present invention is selected as is known in the art. Hairpin ribozymes are now in clinical trial and are the preferred type. In general the ribozyme is from 30-100 nucleotides in length. Modifications or analogues of nucleotides can be introduced to improve the therapeutic properties of the nucleotides. Improved properties include increased nuclease resistance and/or increased ability to permeate cell membranes.
  • Nuclease resistance is provided by any method known in the art that does not interfere with biological activity of the antisense oligodeoxy- nucleotides, cDNA and/or ribozymes as needed for the method of use and delivery (Iyer et al . , 1990; Eckstein, 1985; Spitzer and Eckstein, 1988; Woolf et al . , 1990; Shaw et al., 1991).
  • Modifications that can be made to oligonucleotides in order to enhance nuclease resistance include modifying the phosphorous or oxygen heteroatom in the phosphate backbone. These include preparing methyl phosphonates, phosphorothioates, phosphorodithioates and morpholino oligomers.
  • phosphorothioate bonds linking between the four to six 3 ' -terminus nucleotide bases.
  • phosphorothioate bonds link all the nucleotide bases.
  • Other modifications known in the art can be used where the biological activity is retained, but the stability to nucleases is substantially increased.
  • the present invention also includes all analogues of, or modifications to, an oligonucleotide • of the invention that does not substantially affect the function of the oligonucleotide.
  • the nucleotides can be selected from naturally occurring or synthetic modified bases. Naturally occurring bases include adenine, guanine, cytosine, thymine and uracil.
  • Modified bases of the oligonucleotides include xanthine, hypoxanthine, 2- aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiuracil, 8-halo adenine, 8- aminoadenine , 8-thiol adenine, 8-thiolalkyl adenines, 8- hydroxyl adenine and other 8 -substituted adenines, 8 -halo guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other substituted guanines, other aza and deaza adenines, other aza and deaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytos
  • analogues of nucleotides can be prepared wherein the structure of the nucleotide is fundamentally altered and that are better suited as therapeutic or experimental reagents.
  • An example of a nucleotide analogue is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in DNA (or RNAO is replaced with a polyamide backbone which is similar to that found in peptides.
  • PNA analogues have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vi tro . Further, PNAs have been shown to bind stronger to a complementary DNA sequence than a DNA molecule. This observation is attributed to the lack of charge repulsion between the PNA strand and the DNA strand.
  • Other modifications that can be made to oligonucleotides include polymer backbones, cyclic backbones, or acyclic backbones.
  • the active ingredients of the pharmaceutical composition can include oligonucleotides that are nuclease resistant needed for the practice of the invention or a fragment thereof shown to have the same effect targeted against the appropriate sequence (s) and/or ribozymes.
  • Combinations of active ingredients as disclosed in the present invention can be used including combinations of antisense sequences.
  • the antisense oligonucleotides (and/or ribozymes) and cDNA of the present invention can be synthesized by any method known in the art for ribonucleic or deoxyribonucleic nucleotides. For example, an Applied Biosystems 38OB DNA synthesizer can be used. When fragments are used, two or more such sequences can be synthesized and linked together for use in the present invention.
  • nucleotide sequences of the present invention can be delivered either directly or with viral or non-viral vectors. When delivered directly the sequences are generally rendered nuclease resistant. Alternatively, the sequences can be incorporated into expression cassettes or constructs such that the sequence is expressed in the cell as discussed herein below. Generally, the construct contains the proper regulatory sequence or promoter to allow the sequence to be expressed in the targeted cell.
  • Negative dominant peptide refers to a partial cDNA sequence that encodes for a part of a protein, i.e. a peptide (see Herskowitz, 1987). This peptide can have a different function from the protein it was derived from. It can interact with the full protein and inhibit its activity or it can interact with other proteins and inhibit their activity in response to the full protein. Negative dominant means that the peptide is able to overcome the natural proteins and fully inhibit their activity to give the cell a different characteristics like resistance or sensitization to killing. For therapeutic intervention either the peptide itself is delivered as the active ingredient of a pharmaceutical composition or the cDNA can be delivered to the cell utilizing the same methods as for antisense delivery.
  • gene therapy refers to the transfer of genetic material (e.g. DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition phenotype.
  • the genetic material of interest encodes a product (e.g. a protein, polypeptide, peptide, functional RNA, antisense) whose production in vivo is desired.
  • the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value.
  • the genetic material of interest encodes a suicide gene.
  • ex vivo Two basic approaches to gene therapy have evolved: (1) ex vivo and (2) in vivo gene therapy.
  • ex vivo gene therapy cells are removed from a patient, and while being cultured are treated in vi tro .
  • a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method
  • transfection transduction, homologous recombination, etc.
  • an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient.
  • These genetically reimplanted cells have been shown to express the transfected genetic material in situ.
  • target cells are not removed from the subject rather the genetic material to be transferred is introduced into the cells of the recipient organism in si tu, that is within the recipient.
  • the host gene is defective, the gene is repaired in si tu [Culver, 1998] . These genetically altered cells have been shown to express the transfected genetic material in si tu .
  • the gene expression vehicle is capable of delivery/transfer of heterologous nucleic acid into a host cell.
  • the expression vehicle can include elements to control targeting, expression and transcription of the nucleic acid in a cell selective manner as is known in the art . It should be noted that often the 5 ' UTR and/or 3 ' UTR of the gene can be replaced by the 5 ' UTR and/or 3 ' UTR of the expression vehicle. Therefore as used herein the expression vehicle can, as needed, not include the 5 ' UTR and/or 3 ' UTR of the actual gene to be transferred and only include the specific amino acid coding region.
  • DNA viral vector for introducing and expressing recombinant sequences is the adenovirus derived vector Adenop53TK.
  • This vector expresses a herpes virus thymidine kinase (TK) gene for either positive or negative selection and an expression cassette for desired recombinant sequences.
  • TK herpes virus thymidine kinase
  • This vector can be used to infect cells that have an adenovirus receptor which includes most cancers of epithelial origin as well as others .
  • This vector as well as others that exhibit similar desired functions can be used to treat a mixed population of cells and can include, for example, an in vi tro or ex vivo culture of cells, a tissue or a human subject.
  • viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types.
  • the targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell .
  • the vector to be used in the methods of the invention will depend on desired cell type to be targeted and will be known to those skilled in the art. For example, if breast cancer is to be treated then a vector specific for such epithelial cells would be used. Likewise, if diseases or pathological conditions of the hematopoietic system are to be treated, then a viral vector that is specific for blood cells and their precursors, preferably for the specific type of hematopoietic cell, would be used.
  • Retroviral vectors can be constructed to function either as infectious particles or to undergo only a single initial round of infection.
  • the genome of the virus is modified so that it maintains all the necessary genes, regulatory sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new viral particles which are capable of undergoing further rounds of infection.
  • the vector's genome is also engineered to encode and express the desired recombinant gene.
  • the vector genome is usually mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles. Without such a signal, any particles that are formed will not contain a genome and therefore cannot proceed through subsequent rounds of infection.
  • the specific type of vector will depend upon the intended application.
  • the actual vectors are also known and readily available within the art or can be constructed by one skilled in the art using well-known methodology.
  • the recombinant vector can be administered in several ways. If viral vectors are used, for example, the procedure can take advantage of their target specificity and consequently, do not have to be administered locally at the diseased site. However, local administration can provide a quicker and more effective treatment, administration can also be performed by, for example, intravenous or subcutaneous injection into the subject. Injection of the viral vectors into a spinal fluid can also be used as a mode of administration, especially in the case of neuro-degenerative diseases. Following injection, the viral vectors will circulate until they recognize host cells with the appropriate target specificity for infection.
  • DNA can also be administered using a gene gun.
  • a gene gun Ziao & Brancksman, Nuceic Acids, Res. 24, 2630-2622 (1996) ) .
  • the DNA is precipitated onto the surface of microscopic metal beads.
  • the microprojectiles are accelerated with a shock wave or expanding helium gas, and penetrate tissues to a depth of several cell layers.
  • the AcalTM Gene Delivery Device manufactured by Aegacetus, Inc., Middleton, WI is suitable.
  • nucleic DNA can pass through skin into the bloodstream simply by spotting the DNA onto skin with chemical or mechanical irritation (see WO 95/05853) .
  • non- targeting vectors can be, for example, viral vectors, viral genome, plasmids, phagemids and the like.
  • Transfection vehicles such as liposomes can also be used to introduce the non-viral vectors described above into recipient cells within the inoculated area. Such transfection vehicles are known by one skilled within the art .
  • compositions containing the active ingredients of the present invention as described herein above are administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
  • the pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the medical arts. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the medical arts.
  • the pharmaceutical compositions can be combinations of the active ingredients but will include at least one active ingredient.
  • the pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • the carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) , suitable mixtures thereof, and vegetable oils.
  • Examples of delivery systems useful in the present invention include: 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196.
  • Many other such implants, delivery systems, and modules are well-known to those skilled in the art.
  • a pharmacological formulation of the compound utilized in the present invention can be administered orally to the patient . Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable. Known techniques which deliver it orally or intravenously and retain the biological activity are preferred.
  • the present invention also provides a method of diagnosing the presence of ischemia in a patient including the steps of analyzing a bodily fluid or tissue sample from the patient for the presence or gene product of at least one expressed gene (up-regulated) or their proteins and where ischemia is determined if the up-regulated gene or gene product is ascertained as described herein in the Example.
  • the bodily fluids can include tears, serum, urine, sweat or other bodily fluid where secreted proteins from the tissue that is undergoing an ischemic event can be localized. Additional methods for identification of the gene or gene product are immunoassays, such as and ELISA or radioimmunoassays (RIA) , can be used as are known to those in the art particularly to identify gene products in the samples.
  • Isolated messenger RNA is labeled with fluorescent dNTP's using a reverse transcription reaction to generate a labeled cDNA probe.
  • mRNA is extracted from either C6 or A172 cells cultured in normoxia conditions and labeled with Cy3-dCTP (Amersham) and mRNA extracted from C6 or A172 cells cultured under hypoxia conditions is labeled with Cy5-dCTP (Amersham) .
  • the two labeled cDNA probes are then mixed and hybridized onto microarrays
  • EXAMPLE 1 92 (SEQ ID. No.; 1)
  • a short sequence with features of second paroxysmal targeting signal was found in protein 95 between amino acids 353-361.
  • a putative coiled coil region is found between amino acid positions 253-283.
  • the 95-specific probe that was used for the initial set of hybridizations did not recognize any additional transcripts when hybridized to this newly prepared Northern blot. Therefore, the occurrence of 95-specific additional mRNA species seem to vary among different mRNA preparations . The sizes of PA26 and gene 95 encoded transcripts are different. However, in order to exclude the possibility that the presence of alternative 95 RNA species stem from cross-hybridization, a PA26 specific cDNA probe was synthesized originating from the common exons region. On the Northern blot used for the previous experiment, the PA26 hybridized to two typical mRNA species of 2.6 and 4 Kb. Their levels, unlike the level of 95 mRNA, were not affected by hypoxia.
  • a complete 60F6 human cDNA clone was isolated from A172 cDNA library.
  • the contig is 2675 bp long and contains a single ORF (bp 134 - 866) able to code for a putative protein of 244 amino acids.
  • Rho8 belongs to a family of Ras-related GTPases that regulate the actin cytoskeleton.
  • this protein is unique in that it is constitutively active: GTPase deficient and in vivo farnesylated (Mol Cell Biol . 1996 Jun; 16(6): 2689-99). Therefore, it is intriguing to find that this constitutively active G-protein is regulated on the level of transcription. Hypoxia regulation of Rho8 was not previously described.
  • rat sequence is able to code for protein that represents a rat homologue of human lysyl hydroxylase 2 (PLOD2) .
  • PLOD2 human lysyl hydroxylase 2
  • the full- length open reading frames was cloned for both human and rat lysyl hydroxylase 2 homologues (by PCR, using primers built on the basis of known sequence, for human variant, and degenerative primers, for rat variant) .
  • the encoded proteins have well defined signal peptides .
  • the cloned rat 648 cDNA contains an ORF coding for a putative protein that is 88% identical to the published human PLOD2 sequences. The least conserved sequences are within the signal peptide, however its functional features are completely preserved.
  • the cloned human cDNA is almost identical to published human PLOD2 sequence. The word "almost" in the previous sentence stems from the fact that both in human and in rat cDNA species cloned in QBI a stretch of amino acids between positions 501- 521 of published sequence PLOD2 sequence was absent. Therefore, QBI's PLOD2 variants are differentially spliced. Both rat and human homologues were amplified from RNA extracted from glioma cell lines cultured in hypoxic conditions .
  • Lysyl hydroxylases are the enzymes that catalyze the formation of hydroxylysine in collagens and other proteins with collagen-like amino-acid sequences, by the hydroxylation of lysine residue in X-K-G sequences.
  • the hydroxilysine residues have two important functions: (1) serve as sites of attachment of carbohydrate units, and (2) they are essential for the stability of the intermolecular collagen crosslinks.
  • Congenital deficiency of lysyl hydroxylase in humans leads to increased solubility of collagens and, consequently, to numerous defects in organization of connective tissue in various organs.
  • PLOD2 was found to be highly expressed in pancreas, skeletal muscle, heart and placenta (by Northern blot) . None is known either about the regulation of PLOD2 expression by hypoxia or about its involvement in angiogenesis and tumorigenesis . Induction of PLOD2 by hypoxia can probably account for hypoxia-induced tissue fibrosis. Indeed, specific lysyl hydroxylase inhibitor, minoxidil, was able to suppress both cellular collagen production and fibroblasts proliferation (J. Biol. Chem. , 262, 11973 -8, 1987; Graefes Arch. Clin. Exp . Ophthalmol . 233, 347 -55, 1995) . There were suggestions in literature to use modified lysyl hydroxylase inhibitor for treatment of vitreoretinopathy (Invest. Ophthalmol. Vis .Sci. 34, 567- 75, 1993) .
  • pcDNA3-648 was transiently co-transfected together with pcDNA3-GFP in Hela and 293 cells. 24 and 48 hours later the cells were fixed and stained with DAPI. No apoptotic effect was observed in the transfected cells.
  • a co-transfection assay was conducted using the pcDNA3-GFP and the FAS plasmids. No anti-apoptotic effect was observed.
  • Probe 648 demonstrates clear hybridization signal throughout the inner nuclear layer of "hypoxic” pup's retina while “normoxic” retina is negative for the expression. No hybridization signal was detected in adult retina .
  • hybridization signal was detected in some apoptotic cells in the roof of the fourth brain ventricle and in developing retina ganglia, where expressing cells had no apoptotic features.
  • Multi-tissue block hybridization shows expression of 648 gene (rat PLOD2) in visceral smooth muscles in oviduct, uterus, stomach and intestine. vascular smooth muscles do not display hybridization signal.
  • gene 24D4 is down-regulated after 16 hours of hypoxia. On Northern blots, it appears as a single 1.5 Kb mRNA species.
  • the sequence has no analogs in public databases.
  • the available protein sequence contains three consequent Zn-finger motifs, all of C2H2 type (aa 52-72, 80-100 and 108-128).
  • Zinc finger domains of this type are usually found in nucleic acid-binding proteins.
  • EST clone 18E contains 580 bp, that include an ORF for a 120 aa protein (bp 92 - 452) .
  • the EST clone 3D contains 486 bp.
  • Comparison of nucleotide sequences of clone 18E and 3D reveal that the latter has a out-of-frame deletion of 71 nucleotides between bp 279-339 of clone 18E (its putative coding region) (Fig.68) . This raised the question whether 77H4 cDNA is at all coding. The sequence was analyzed by Genscan program, that predicts the potential coding sequences on the basis of codon usage.
  • the 77H4 cDNA clone has similar to SRA function and can serve a coactivator in some transcriptional complexes.
  • gene 14G2 is regulated within 16 hours of hypoxia. On Northern blots, it appears as a single mRNA species .
  • gene 29F3 is regulated within 16 hours of hypoxia. On Northern blots, it appears as a single mRNA species.
  • Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat Med. 1(10) :1024-1028.
  • VEGF vascular endothelial growth factor
  • Pet-1 a novel ETS domain factor that can activate neuronal nAchR gene transcription. J Neurobiol. 34 (2) : 151-163.
  • Tumor cell autocrine motility factor is the neuroleukin/phosphohexose isomerase polypeptide. Cancer Res. 56 (13) :2960-2963.
  • Huxley et al . "The human HPRT gene on a yeast artificial chromosome is functional when transferred to mouse cells by cell fusion", Genomics , 9:742-750 (1991). Jakobovits et al . , "Germ-line transmission and expression of a human-derived yeast artificial chromosome", Nature, Vol. 362, pp. 255-261 (1993) . Lamb et al . , "Introduction and expression of the 400 kilobase precursor amyloid protein gene in transgenic mice", Nature Genetics, Vol. 5, pp. 22-29 (1993).

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L'invention concerne des séquences de polynucléotides utilisées pour identifier des gènes modulés par des conditions hypoxiques.
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US9713723B2 (en) 1996-01-11 2017-07-25 Impulse Dynamics Nv Signal delivery through the right ventricular septum
EP1002862A1 (fr) * 1998-11-12 2000-05-24 Nitsch, Roger M., Prof. Dr. Procédés pour diagnostiquer ou traiter des maladies neurologiques
US9101765B2 (en) 1999-03-05 2015-08-11 Metacure Limited Non-immediate effects of therapy
WO2002044419A2 (fr) * 2000-11-28 2002-06-06 Wyeth Analyse d'expression d'acides nucleiques kiaa et polypeptides utilises dans le diagnostic et le traitement du cancer de la prostate
EP1398378A4 (fr) * 2001-05-31 2005-03-30 Chiba Prefecture Acides nucleiques isoles dans le neuroblastome
EP1410013A4 (fr) 2001-06-21 2006-02-01 Millennium Pharm Inc Compositions, necessaires et methodes destines a l'identification, a l'evaluation, a la prevention et au traitement des cancers du sein et de l'ovaire
EP1578367A4 (fr) * 2002-11-01 2012-05-02 Genentech Inc Compositions et methodes pour le traitement de maladies liees au systeme immunitaire
US11439815B2 (en) 2003-03-10 2022-09-13 Impulse Dynamics Nv Protein activity modification
US20080249038A1 (en) * 2003-10-07 2008-10-09 Quark Biotech, Inc. Bone Morphogenetic Protein (Bmp) 2A and Uses Thereof
US8548583B2 (en) 2004-03-10 2013-10-01 Impulse Dynamics Nv Protein activity modification
US11779768B2 (en) 2004-03-10 2023-10-10 Impulse Dynamics Nv Protein activity modification
CA2594673A1 (fr) 2004-12-09 2006-07-13 Impulse Dynamics Nv Modification de l'activite proteique
US8934975B2 (en) 2010-02-01 2015-01-13 Metacure Limited Gastrointestinal electrical therapy
JP2011206049A (ja) * 2010-03-08 2011-10-20 Sumio Sugano 壊死マーカー及びその用途
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