WO1996010401A1 - Procedes de regulation du recepteur du facteur de croissance de type 1 proche de l'insuline - Google Patents

Procedes de regulation du recepteur du facteur de croissance de type 1 proche de l'insuline Download PDF

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WO1996010401A1
WO1996010401A1 PCT/US1995/012563 US9512563W WO9610401A1 WO 1996010401 A1 WO1996010401 A1 WO 1996010401A1 US 9512563 W US9512563 W US 9512563W WO 9610401 A1 WO9610401 A1 WO 9610401A1
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Patrick Delafontaine
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Emory University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/13—Decoys
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • the present invention relates to the use of oligonucleotide-mediated methods for the regulation of growth factor receptor gene expression, particularly insulin-like growth factor 1 receptor, and methods for wound healing, promoting blood vessel proliferation, preventing postangioplasty restenosis and/or post transplant atherosclerosis, and regulating smooth muscle cell growth.
  • IGF I Insulin-like growth factor I
  • IGF 1 which is synthesized and secreted by vascular smooth muscle cells, acts as an autocrine/paracrine factor. IGF 1 acts to stimulate progression of cells from the Gl phase to the S phase of the cell cycle. Thus, IGF 1 serves a critical role in cell growth and proliferation and in normal development.
  • IGF insulin growth factor
  • IGF IR membrane tyrosine kinase
  • the IGR IR is a glycosylated heterotetramer which consists of two extracellular ⁇ subunits and two transmembrane ⁇ subunits which each have extracellular and intracellular portions. The or subunits contain the growth factor binding site.
  • growth factors including platelet-derived growth factor (PDGF) , basic fibroblast growth factor (bFGF) , and Angiotensin II (Ang II) upregulate vascular smooth muscle cell (VSMC) IGF IR (Pfeile et al. (1987) Endocrinology, 120.2251-2258: Ververis et al.
  • Angiotensin II (Ang II) , a vasoactive and mitogenic peptide, transcriptionally regulates the IGF I gene in VSMC, and neutralization of extracellular IGF I with an anti-IGF I antibody inhibits ang II-induced DNA synthesis in VSMC (Delafontaine et al. (1993) J. Biol . Chem. 268:16866-16890) ⁇
  • the present invention provides methods for regulating cell growth, in particular vascular smooth muscle cell growth, and methods for stimulating proliferative process including, but not limited to, wound healing, burn healing, bone healing, nerve regeneration, and angiogenesis.
  • Negative regulation of vascular smooth muscle cell growth and proliferation allows for the prevention or treatment of medical conditions including, but not limited to, coronary artery disease, post-angioplast restenosis, post coronary transplant atherosclerosis, atherosclerotic lesions and vascular complications of hypertension, including hypertensive retinopathy, hypertensive nephropathy, cerebrovascular disease and myocardial hypertrophy, and in the treatment of solid cancers.
  • Negative regulation of cell proliferation in particular of vascular smooth muscle cells is achieved using antisense oligonucleotides, which oligonucleotides are at least about 14 nucleotides, and preferably about 18 to about 22 nucleotides, in length and comprises contiguous sequence complementary to at least a portion of the mRNA encoding a growth factor receptor, for example, type I insulin-like growth factor receptor (IGF IR) .
  • the antisense oligonucleotide is complementary to the growth factor receptor mRNA sequence in the vicinity of the translation start site of the coding sequences.
  • other antisense sequences complementary to coding sequence information is effective for inhibiting factor receptor gene expression, thereby inhibiting target cell growth and proliferation.
  • Antisense nucleic acid molecules can also be made as transcription products from non-naturally occurring recombinant nucleic acid molecules expressed in the target tissue.
  • expressed antisense RNAs are at least about 100 nucleotides in length and up to 1 Kb or more.
  • IGF IR antisense oligonucleotides useful in the present methods include those having sequences as given in SEQ ID NO:l (ATG-directed) and SEQ ID NO: 4.
  • IGF IR mRNA effectively inhibit IGF IR expression in rat and human cells, such as vascular smooth muscle cells, thereby preventing or ameliorating post angioplasty restenosis and post-transplant atherosclerosis of the coronary arteries, and inhibiting angiogenesis in solid tumors.
  • Another aspect of the present invention is the positive regulation of growth factor receptor gene expression via ATG- directed sense oligonucleotides and, as specifically embodied herein, for up-regulation of type I insulin-like growth factor receptor gene expression, with the result that cell growth and proliferation is stimulated.
  • ATG-directed oligonucleotides are applied to or expressed in target tissue
  • the target tissue can be wounded or burn*.
  • angiogenesis development of collateral blood vessels
  • ATG-directed sense oligonucleotides effective for stimulating growth factor receptor gene expression and, as specifically exemplified, an oligonucleotide comprising a sequence as given in SEQ ID NO: 2 is effective in human and rat cells and tissue.
  • Functionally equivalent ATG-directed sense oligonucleotides can be readily produced, without the expense of undue experimentation, for other species using the teachings of the present disclosure taken with knowledge and techniques readily accessible to the art.
  • the antisense oligonucleotides can be chemically synthesized from ribonucleotide or deoxynucleotide precursors or they may be transcription products expressed from non-naturally occurring recombinant DNA molecules such as adenovirus or Epstein-Barr virus vectors. Preferably, such vectors do not persist in the tissues for very extended periods of time.
  • Preferred chemically synthesized sense or antisense oligonucleotides are those with chemical modifications which prolong persistence of intact molecules inside and outside cells, including, but not limited to, phosphorothioate, phosphorodithioate, phosphotriester, methylphosphonate, phosphoramidite, ⁇ -anomer and phosphoroselenoate oligonucleotides, the syntheses of which are known to the art.
  • Figure 1 illustrates the effects of IGF IR ATG-directed oligonucleotides on basal and serum-induced DNA synthesis.
  • VSMC were incubated in serum-free medium alone (SFM) or in the presence of antisense (AS) , mismatch (M) , or sense (S) oligonucleotides (SEQ ID Nos:l, 3 and 2, respectively) (0.1-10 uM) for 48 hr.
  • [ 3 H] -Thymidine incorporation was then determined as basal levels (in the continued presence of SFM) and in response to 10% calf serum (10% CS) as described in Example 3.
  • Results are the mean ⁇ standard error (SE) of duplicate determinations from three to ten independent experiments for each condition.
  • SE standard error
  • Figure 2 illustrates the effects of IGF IR ATG-directed oligonucleotides on the proliferation of VSMC.
  • 50% confluent VSMC were exposed to SFM, DMEM with 10% CS alone, or DMEM with 10% CS and increasing concentrations of AS, M or S oligonucleotides (SEQ ID NOS:l, 3 and 2, respectively) (0.1 - 10 uM) .
  • Cell counts were determined at 96 hr. Shown is the mean ⁇ SE of duplicate measurements from four separate experiments.
  • FIG 3 illustrates the effects of IGF IR oligonucleotides on the mitogenic response to IGF I.
  • VSMC were incubated for 48 hr in DMEM containing 10% CS alone or in the presence of 5 uM AS or S oligonucleotides (SEQ ID NOS: 1 and 2, respectively) .
  • Cells were . then exposed to fresh SFM containing increasing concentrations of human recombinant IGF I (0 - 50 ng/ml) for 24 hr and [ 3 H] -thymidine incorporation determined as described in Example 3. Results are the mean ⁇ SE of duplicate determinations from four separate experiments.
  • Figure 4 illustrates the effects of IGF IR oligonucleotides on mitogenic response to ang II.
  • VSMC were incubated for 48 hr in DMEM containing 10% CS alone or in the presence of 5 uM AS, M or S oligonucleotides (SEQ ID NOS: 1, 3 and 2, respectively) . Cells were then exposed to fresh SFM containing increasing concentrations of ang II (0 - 1000 nM) for 36 hr. [ 3 H] -thymidine incorporation was determined as described in Example 3. Results are the mean ⁇ SE of duplicate measurements from three to seven experiments for each condition.
  • FIGS 5A-5C illustrate the effects of ATG-directed IGF IR oligonucleotides on ICF IR and Angiotensin II (Ang II) receptor numbers.
  • vascular smooth muscle cells VSMC were incubated in
  • Figures 6A-6B illustrate the effects of non-ATG-directed sense (S) and antisense (AS) oligonucleotides (SEQ ID Nos. 5 and 4, respectively) on VSMC DNA synthesis and IGF IR number.
  • S non-ATG-directed sense
  • AS antisense
  • VSMC were incubated in SFM alone or in the presence of AS or S oligonucleotides (0.1 - 10 ⁇ M) for 48 hours.
  • [ 3 H] - thymidine incorporation was then determined basally (in the continued presence of SFM) or in response to 10% CS as described in Example 6. Results are the mean ⁇ standard error of duplicate measurements from three to eight experiments for each condition.
  • Fig. 6A VSMC were incubated in SFM alone or in the presence of AS or S oligonucleotides (0.1 - 10 ⁇ M) for 48 hours.
  • [ 3 H] - thymidine incorporation was then determined basally (in the continued presence of S
  • Figure 7 illustrates the effects of ATG-directed IGF IR antisense (AS) , sense (S) and mismatch (M) oligonucleotides on IGF IR mRNA levels.
  • AS ATG-directed IGF IR antisense
  • S sense
  • M mismatch
  • VSMC were incubated for 48 hours in 10% CS alone (control) or in the presence of 5 ⁇ M AS, M or S ODNs.
  • Total RNA was extracted and co-hybridized using [ 32 P] -UTP labeled IGF IR and GAPDH antisense riboprobes.
  • Figure 8 diagrammatically illustrates the construction of episomal vector pAnti-IGF IR.
  • the rat IGF IR cDNA clone pll8 was digested by Xhol and Kpnl and the 0.8 kb restriction fragment was ligated in an antisense orientation into pCEP4.
  • CMV cytomegalovirus immediate early gene enhancer/promoter
  • SVpA simian virus 40 poly A
  • EBNA-1 EBV-encoded nuclear antigen 1 / oriP, EBV origin of replication
  • Hyg hygromycin resistance gene
  • Amp ampicillin resistance gene
  • Km kanamycin resistance gene
  • Figure 9A-9B illustrate the effect of antisense IGF IR cDNA transcription on IGF IR number and on DNA synthesis.
  • Figure 9A shows IGF IR number per 10 5 cells in CA9 antisense-expressing and control cells. Confluent and up to two days postconfluent monolayers of clone CA9 (transfected with pAnti-IGF IR, CA) and of clones ME8 and ME10 (transfected with vector alone, ME) underwent 125 I-IGF I radioligand binding studies as described in Example 6 herein. Binding data were analyzed using the LIGAND program.
  • FIG. 10 illustrates the effect of antisense IGF IR cDNA transcription on VSMC proliferation. Growth of cells of clone CA9 (transfected with pAnti-IGF IR) and growth of cells of clone ME10 (transfected with vector alone) are compared. Cells were seeded at a density of 2 x 10 3 cells/well and maintained in culture medium containing 10% calf serum. The results shown are representative of an experiment which was repeated three times.
  • Type I insulin-like growth factor (IGF I) is a ubiquitous peptide that circulates at high levels in serum and is expressed in multiple tissues. It has a broad spectrum of effects including stimulation of embryonic and postnatal growth, cell growth and differentiation in vi tro, various metabolic effects and participation in tissue regeneration. IGF I also participates in the undesirable proliferation of vascular smooth muscle cells, e.g., associated with post-angioplasty restenosis, restenosis after other vascular injury and with post-cardiac transplant atherosclerosis. The structure of IGF I is known, and the structure and recombinant expression of human IGF I are described in, e.g., U.S. Patent No. 5,324,639 (Brierly et al) and in Gritz and Davies (1983) J. Gene 5:179-L ' .
  • IGF I The effects of IGF I are mediated via ⁇ ..iding to a specific heterotetrameric membrane receptor (IGF IR) that consists of two extracellular ⁇ -subunits and two membrane-spanning ⁇ -subunits, which receptor is widely distributed in mammalian tissues.
  • IGF IR heterotetrameric membrane receptor
  • SEQ ID NO:10 presents the human IGF IR cDNA sequence and predicted amino acid sequence.
  • the N-termini of the a and the ⁇ subunits are at amino acids 1 and 711, respectively.
  • Potential N-linked glycosylation sites are at amino acids 21-23, 72-74, 105-107, 214-216, 284-286, 397-399, 408-410, 504-506, 577-579, 592-594, 610-612, 726-728, 734-736, 870-872, 883-885 and 933-935.
  • the transmembrane domain is at amino acids 906-929.
  • a potential ATP binding*site is at Gly residues at amino acid numbers 976, 978 and 981 and at Lys 1003.
  • the putative precursor processing site is at 707-710, proteolytic cleavage here divides the precursor into the a and ⁇ subunits.
  • IGF I is a smooth muscle cell mitogen (Pfeifle et al (1982) Horm. Metab. Res . 14:409-414; Clemmons, D.R. (1985) Endocrinology 117:77-83; Clemmons and Van Wyk (1985) J " . Clin . Invest . 5:1914-1918) and several reports have documented that vascular smooth muscle cells (VSMC) express IGF I and its receptor in vi tro and in vivo (See, e.g., Jialal et al (1985) Endocrinology 117:1222-1229; King et al.
  • VSMC vascular smooth muscle cells
  • Angiotensin II (Ang II) , a vascular peptide that is a VSMC mitogen, transcriptionally regulates the IGF I gene in VSMC, and neutralization of extracellular IGF I abolishes Ang II- induced growth.
  • Growth factors such as Ang II, platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) increase IGF I receptors on VSMC, suggesting that upregulation of IGF I receptors may play a role in growth factor induced mitogenic responses.
  • an antisense oligonucleotide is one which has a nucleotide sequence that is complementary to a particular target nucleic acid molecule, usually a mRNA.
  • an antisense is preferably from about 14 nucleotides in length up to the entire target sequence. Oligonucleotides which are chemically synthesized are preferably about 18-22 nucleotides in length.
  • the antisense molecule is an antisense RNA, i.e., a transcription product within a target cell
  • the antisense RNA is at least about 100 nucleotides long, and may be as long as 1 Kb or larger. It is generally preferred that the nucleotide sequence of the antisense oligonucleotide is targeted to the region of the mRNA which includes the translation start site.
  • a sense oligonucleotide is one which has a sequence identical to at least a portion of a target nucleic acid molecule, usually a target mRNA.
  • a sense oligonucleotide introduced into or expressed in a cell may complex with a naturally occurring target nucleic acid molecule, or which significantly differs in overall nucleotide sequence (by more than about 45%) from a molecule of the same sense polarity.
  • an ATG-directed oligonucleotide can be sense or antisense relative to a target mRNA.
  • ATG-directed means that the oligonucleotide comprises the ATG or AUG translation start site in a sense oligonucleotide, or its complement CAT (or CAU in RNA) in the antisense oligonucleotide, and the limitations on length and possibly advantageous modifications described herein.
  • An ATG-directed sense of antisense RNA (a less than full length transcript specific for IGF IR or other growth factor receptors) similarly comprises the ATG (or AUG) translation start site or its CAT (or CAU in RNA) complement, depending on whether it is sense or antisense, and with length limitations as described herein.
  • a mismatch (or missense) antisense oligonucleotide is one which has an antisense nucleotide sequence in which there is significant mismatch with a complementary target nucleic acid molecule.
  • an oligonucleotide comprising chemical modifications which contribute to chemical stability, resistance to enzymatic degradation- and the like.
  • chemically modified oligonucleotides include, but are not limited to, phosphorothioate (e.g., 2-O-methylphosphorothioate) , phosphorodithioate, phosphotriester, methylphosphonate, phosphora idite, ⁇ -anomer and phosphoroselenoate oligonucleotides.
  • Phosphorothioate (sulfur-protected) oligonucleotides are relatively stable in serum, with about 50% remaining intact after 36 hours incubation, according to Shi et al (1993) Circulation 8.:1190-1195.
  • This reference describes uptake by and persistence in and inhibition of human smooth muscle cell proliferation by antisense phosphorothioate oligonucleotides directed against c_z. myc messenger RNA.
  • DNA or RNA is one which does not occur in nature, and it is thus distinguished from a chromosome, for example.
  • a specific example of a recombinant DNA molecule of the present invention is a vector comprising a portion of an IGF IR sequences operably linked to expression control sequences such that transcriptional expression of at least a portion of the IGF IR sequences is obtained.
  • Those expression control seq - nces regulate and promote transcription, and they are not associated with IGF IR coding sequences in nature.
  • the transcription product will be sense or antisense, as understood in the art.
  • the transcript is an antisense RNA and is complementary to at least about 100 nucleotides of the native cellular IGF IR mR' A.
  • the expression product is a sense RNA which is sh ter than native cellular IGF IR nRNA, does not contain a : -1-length coding sequence, but which include 1- the region of the ATG translation* stare site.
  • he sense RNA is significantly shorter than ti full i ,th coding sequence, e.g., about 100 nucleotides in len.-h.
  • An additional embodiment is a vector in which a vertebra t e IGF IR cDNA sequence with full length coding sequence (e rat) has been inserted, operably linked to expression contrcx sequences.
  • Vectors may be plasmids or virus vectors capable of being maintained in bacterial cells, yeast cells or in vertebrate, such as mammalian cell culture. These vectors may be designed for maintenance in a single-celled host (bacterium or yeast) and capable only of transient gene expression when introduced into a human, or other animal or capable of stable maintenance with either regulated (temporally and/or tissue-specific) or constitutive gene expression.
  • the art knows how to choose vectors and expression control sequences to achieve a particular desired result from a number of known and readily accessible vectors and expression control sequences known to the art.
  • transient expression is preferred.
  • antisense RNA expression in treatment and/or prevention of atherosclerosis, post balloon angioplasty restenosis and post transplant atherosclerosis and vascular smooth muscle cell hyperproliferative disorders transient expression of antisense RNA is preferred.
  • antisense oligonucleotides can be substituted.
  • IGF IR gene expression In applications of stimulating IGF IR gene expression, such as in the production of leaner meat in, e.g., swine or beef cattle, prolonged expression of a relatively short (at least 14-20 nucleotides, but less than full length) sense ATG-directed IGF IR RNA is preferred.
  • the cDNA sequence for rat brain cell IGF IR was determined from several overlapping clones isolated as described in Example 1. Nucleotide sequence analysis of the cDNA resulted in deduction of the primary protein sequence of the rat IGF IR precursor (SEQ ID NOS: 8 and 9) .
  • the cDNA analyzed contains an opening reading frame of 4113 nucleotides encoding a 1370 amino acid IGF IR precursor, with 45 bp of 5' untranslated and 538 bp of 3' untranslated flanking sequences.
  • the first 30 encoded amino acids likely represent a signal peptide (amino acids -30 to -1) with the putative start site of the mature subunit being glutamate at position 1 (SEQ ID NO: 9) .
  • SEQ ID NO: 9 Four arginine residues representing the putative cleavage site of the ⁇ proreceptor precede the putative start site of the ⁇ subunit at Asp 712.
  • the 17 amino acid hydrophobic sequence between residues 908 and 924 is likely a transmembrane domain.
  • Features consistent with kinase activity are located between amino acids 974 and 1230, including the tyrosine kinase class II signature pattern from Asp 1130 to Arg 1138 and a potential ATP-binding region from Leu 976 to Val 984.
  • Lammers et al. (1989) EMBO J. 8.:1369-1375 describes a recombinant vector for the expression of human IGF IR.
  • the predicted rat ⁇ -chain comprises 707 amino acids
  • the ⁇ -chain is highly conserved between rat and human with 90% and 98% similarity at nucleotide and amino acid levels.
  • a 17 amino acid hydrophobic sequence (residues 908 to 924) likely represents the transmembrane domain, and differs from the corresponding human sequence by the substitution of an isoleucine for valine at position 915.
  • the highly conserved tyrosine kinase domain contains a protein kinase ATP binding signature (Leu 976 to Val 1084) , a tyrosine kinase specific active-site signature (Phe 1102 to Val 1114), and a receptor tyrosine kinase class II signature
  • SEQ ID NO:2 increased cell number (30% increase with 10 ⁇ M S ODN over 10% calf serum alone) .
  • IGF I-induced growth (mitogenic) response cells were incubated in 10% calf serum ⁇ oligonucleotides (ODNs) for 48 hr and then exposed to serum-free medium in the presence or absence of increasing concentrations of IGF I (Fig. 3) .
  • IGF I caused a dose-dependent increase in [ 3 H] -thymidine incorporation that was markedly blunted by pre-incubation of cells with ATG-directed AS ODNs.
  • [ 3 H] -thymidine incorporation stimulated by 20 ng/ml IGF I was reduced by 62% in AS-treated cells, compared with control.
  • Exposure of cells to 5 ⁇ M S ODN increased [ 3 H] -thymidine incorporation basally (187% above control) and in response to IGF I (67% increase above control following incubation with 20 ng/ml IGF I) .
  • an antisense IGF IR construct was stably transfected into primary rat aortic smooth muscle cells (RASM) .
  • RASM primary rat aortic smooth muscle cells
  • antisense-expressing cells had a 59% decrease in IGF I-stimulated DNA analysis. These findings were substantiated by growth curves which showed a marked reduction in the proliferation of CA9 cells maintained in 10% serum. Interestingly, there was only a small decrease in IGF IR mRNA levels in antisense-expressing cells at preconfluence, correlating with a 35% reduction in IGF IR number. The lesser reduction in IGF IR mRNA in preconfluent cells may relate to the fact that preconfluent control cells have approximately 1.5-fold higher basal IGF IR mRNA levels than post-confluent cells. The greater reduction in IGF IR at confluency in antisense cells correlates with a marked reduction in cellular growth rates.
  • IGF IR-related insert IGF IR-related insert
  • solution hybridization/RNase protection assays were performed using an antisense IGF IR riboprobe, co-hybridized with an antisense GAPDH riboprobe as an internal control. There was a small reduction in IGF IR mRNA levels in preconfluent cells and marked reduction 5 in post-confluent cells.
  • Anti-IGF I antiserum has been shown to inhibit PDGF-mediated growth of VSMC (Clemmons and Van Wyk (1985) supra) . Delafontaine and Lou (1993) supra showed that Ang II transcriptionally regulated IGF I expression in VSMC and that anti-IGF I antibody inhibits Ang II induced DNA synthesis. In neuroepithelial cells FGF-mediated proliferation is dependent on IGF I (Drago et al. (1991) Proc . Nat 'l Acad. Sci . USA 8:2199- 2203) . There is some experimental suggestion of the importance of IGF IR number/cell in cellular growth responses.
  • Antisense targeting of the IGF I/IGF IR system has provided evidence for its role in normal organ growth (Wada et al. (1993) Proc . Nat 'l . Acad. Sci . USA £0 . :10360-10364) and in tumorigenesis (Trojan et al. (1992) Proc . Nat 'l . Acad. Sci . USA 819:4874-4878) . Furthermore, overexpression of the IGF IR has been shown to induce transformation of NIH/3T3 cells in vi tro, allowing these cells to form tumors in nude mice (Kaleko et al. (1990) Mol . Cell . Biol . 3-0.:464-473) .
  • IGF IR plays a crucial role in VSMC proliferative responses and provides the basis for antisense methods to down-regulate IGF IR gene expression to 19 retard VSMC growth and proliferate where undesirable, e.g., in treatments to prevent post angioplasty restenosis, restenosis after vascular injury or post-heart-transplant atherosclerosis in the coronary arteries and vascular complications of hypertension includingproliferative retinopathy, cerebrovascular disease and hypertensive nephropathy. And, on the contrary, ATG- directed sense oligonucleotides and methods using same allow for stimulation of cell growth and proliferation where desirable. Such situations where increased IGF IR number/cell is desirable include healing of broken bones, burns and other wounds, nerve regeneration after injury, and angiogenesis in tissue affected by peripheral vascular disease or by myocardial infarction.
  • IGF IR density on vascular smooth muscle cells is an important determinant of their growth responses to serum and to angiotensin II.
  • targeting of the IGF IR mRNA with antisense ODNs complementary thereto as exemplified with either a sequence spanning the translation initiation codon or the sequence beginning about 109 bp downstream thereof, reduces IGF IR mRNA levels and IGF IR number without altering IGF IR binding-affinity
  • the mitogenic response to ang II was also inhibited by antisense oligonucleotides complementary to IGF IR mRNA, confirming the crucial role of the autocrine IGF I ligand-receptor system in ang II-induced growth of VSMC. It was previously demonstrated that an anti-IGF I antibody inhibits ang Il-induced mitogenesis in VSMC (Delafontaine et al. (1993) supra) . The effect of IGF IR antisense oligonucleotides on ang II mitogenesis was specific to IGF IR gene expression, in that ang II binding capacity was not altered.
  • an ATG-directed mismatched antisense oligonucleotide (based on the antisense sequence of SEQ ID NO:l but with a 9 of 20 bp mismatch) had no effect on IGF I receptor number, [ 3 H] -thymidine incorporation (basally or in response to serum) , cell proliferation and the mitogenic response to ang II.
  • VSMC exposure of VSMC to a sense oligonucleotide corresponding to sequence spanning the initiation codon of the IGF IR (e.g., S, SEQ ID NO:2) resulted in an increase in IGF IR number per cell, associated with an increase in basal [ 3 H] - thymidine incorporation (in serum-free medium) as well as in the presence of 10% serum. This was reflected in an increase in cell proliferation.
  • the increase in IGF IR density induced by S ODN of SEQ ID NO:2 was associated with an increase in the mitogenic response to IGF I.
  • ang II had no additive stimulatory effect on [ 3 H] -thymidine incorporation following pre-incubation of cells with S ODN (SEQ ID NO:2) . This suggests that at a certain level of activation of the IGF IR, angiotensin II does not further stimulate mitogenesis.
  • IGF IR antisense mRNA species in VSMC, which is effectively "neutralized” by the administered sense oligonucleotide.
  • reports of naturally occurring antisense RNAs in eukaryotic systems are rare (Inouye, M. (1988) Gene T ⁇ _ : 2 S - _ ⁇ _ ; Colman, A. (1990) J. Cell . Science 97:399-409: Helene et al. (1990) Biochem. Biophys . Acta . 1049:99-125) .
  • the S mRNA may bind to its complementary DNA sequence through Watson-Crick bonding or through triple helix formation and increase transcription by facilitating the opening of the DNA helix.
  • local openings of the double helix have been shown to correlate with DNA template activity, and clearly, epigenetic RNA molecules are capable of stabilizing these openings (Reiss et al. (1991) Cancer Research 51:5997-6000.
  • a third non-binding potential explanation is that the ATG- directed sense oligonucleotide may bind with a protein factor to effect positive regulation of IGF IR gene expression.
  • EGF EGF upregulates IGF I expression; and secretion and targeting of the IGF IR through use of antisense oligonucleotides inhibits
  • IGF I-mediated growth occurs independently of the EGF and PDGF receptors (Pietrzkowski et al . (1992) Cell Growth Differ. 3 . :199-205) .
  • IGF IR density is an important factor in mediating IGF I and serum- induced growth responses in vascular smooth muscle cells.
  • the mitogenic response of antisense-treated cells is markedly blunted.
  • IGF I acting through its tyrosine-kinase receptor, may serve as an important co-factor or intermediary in the growth response to a variety of agonists. This is consistent with its known effects at the G-j/S phase of the cell cycle (Stiles et al. (1979) Proc . Natl . Acad. Sci USA 26_:1279-1283) .
  • IGF IR density On vascular smooth muscle cells markedly alters the growth responses of these cells to IGF I, ang II and serum. Receptor availability has marked effects on ligand responsiveness. This ligand-receptor system is crucial for the control of VSMC growth in vitro and .in vivo. Furthermore; the surprising observation that a sense oligonucleotide targeting of the ATG site of the IGF IR message upregulates IGF IR identifies a novel use for synthetic oligonucleotides in the regulation of gene expression.
  • the density of IGF IR can be down- regulated via antisense oligonucleotides or antisense RNAs complementary to at least a portion of the IGF IR mRNA ( ⁇ 14 contiguous nucleotides for oligonucleotides, at least 100 nucleotides for intracellularly transcribed antisense RNAs) .
  • Antisense oligonucleotides and antisense RNAs inhibit vascular smooth muscle cells in vitro and have the same effect .in vivo.
  • oligonucleotides when they are used, they are in a modified form which increases persistence in cells and/or in the extracellular milieu.
  • An exemplified modified modification is phosphorothioate oligonucleotides.
  • ATG-directed sense oligonucleotides e.g., phosphorothioate oligonucleotides
  • IGF IR mRNA can be delivered when the proliferation of vascular smooth muscle cells, or when collateral blood vessel development is desired.
  • a catheter can be used or a porous balloon generally similar to that used in balloon angioplasty can be used (See, e.g., Shi et al. (1994) Circulation jK):944-951; Guzman et al. (1993) Circulation :2838-2848; Ohno et al (1994) Science 265:781-784) .
  • ATG-directed, IGF IR-specific oligonucleotides preferably modified for resistance to degradation (e.g. , in phosphorothioate form)
  • pharmaceutical preparations of IGF IR-specific, ATG-directed sense oligonucleotides, preferably a modified oligonucleotide such as a phosphorothioate oligonucleotide can be formulated, in a therapeutically effective amount, with a suitable carrier for topical application or other local application such as introduction via catheter.
  • An adenovirus vector capable of expressing an IGF IR-specific, ATG-directed sense RNA can also be formulated into a healing-promoting preparation.
  • a vector is replication-deficient.
  • an IGF IR-specific antisense oligonucleotide such as a phosphorothioate oligonucleotide
  • an antisense-expressing recombinant vector can be incorporated.
  • an IGF IR-specific antisense-expressing recombinant vector or oligonucleotide preparation can be directed to a target tissue such as a solid tumor using a means known to the art.
  • a target tissue such as a solid tumor using a means known to the art.
  • Suitable excipients for therapeutic/pharmaceutical compositions are described in, e.g., Remington's Pharmaceutical Sciences, by E.W. Martin.
  • Other components such as stabilizers including chelating agents, e.g., EDTA, sugars, sugar alcohols, antioxidants and nonionic surfactants such as polyethylene glycol or block co-polymers of polyethylene and polypropylene glycol may be added.
  • Therapeutic oligonucleotide (or vector) compositions of the present invention contain a therapeutically effective dose of the active ingredient in a pharmaceutically acceptable carrier.
  • the dose, carrier, route and schedule of administration of choice 24 depend among other factors, upon each other, the condition to be treated and the patient's species. These factors are readily determined and monitored by the treating physician or veterinarian during the course of therapy. All references cited herein are hereby incorporated by reference in their entirety.
  • Primers Dl (5' -CCCCAAATAAAAGGAATG-3) (SEQ ID NO:6) and Ul (5'- AGCAGATTGCCCTTC-3' ) (SEQ ID NO:7) were based on the sequence of Werner et al. (1989) Proc. Nat ' l Acad. Sci . USA 86 . : 7451-7455.
  • the PCR product was directly cloned into the pCRII vector (Invitrogen Inc., San Diego, CA) , and the resulting clone (pll ⁇ ) was sequenced and used to screen a ⁇ gtlO cDNA library from adult male Sprague-Dawley rat brain (Clontech, Palo Alto, CA) .
  • 1.2 x 10 6 clones were screened, and 30 of 80 positive clones were analyzed by PCR using a ⁇ gtlO arm universal primer and an IGF IR internal primer to determine insert size.
  • clones containing longer inserts were isolated, purified and the library was rescreened using these clones and a 3' specific probe (from 2887-2906 bases downstream of the IGF IR translation start site) .
  • EcoRI inserts of purified phage clones were subcloned into pGEM3 (Promega Corp., Madison, WI) and sequenced using the sequenase version 2.0 kit (United States Biochemical Corp., Cleveland, OH) .
  • Ambiguities were resolved by resequencing using a modified 40% formamide gel (J.T. Baker, Inc., Phillipsburg, NJ) .
  • DNA sequence data was assembled from nine overlapping clones and analyzed using the Genetics Computer Group program (Madison, WI) .
  • the deduced amino acid sequence data was analyzed using PC Gene (IntelliGenetics, Inc., Mountain View, CA) .
  • PC Gene IntelliGenetics, Inc., Mountain View, CA
  • a full-length rat cDNA clone for IGF IR was also isolated.
  • Phosphorothioate 20-mer oligonucleotides synthesized using an Applied Biosystems instrument (Applied Biosystems, Foster City, CA) by the Microchemical Facility, Emory University, HPLC purified, resuspended in TE (10 mM Tris, 1 mM EDTA; pH 8.0) , and quantified by spectrophotometry.
  • Antisense (AS) , sense (S) and mismatch (M) ODNs targeting a sequence starting 2 nucleotides 5' to (upstream of) the ATG site of rat IGF IR cDNA were respectively; AS, 5' -TCCGGAGCCAGACTTCATTC-3' (SEQ ID NO:1), S, 5'- GAATGAAGTCTGGCTCCGGA-3 ' (SEQ ID NO:2) ; M, 5'- AGCGGTCCCACTCTTGTTTG-3' (SEQ ID NO:3) .
  • ODNs targeting a sequence starting 109 bases downstream of the ATG were AS, 5'- CAGCTGCTGATAGTCGTTGC (SEQ ID NO:4) and S, 5'- GCAACGACTATCAGCAGCTG-3' (SEQ ID NO:5) .
  • Oligonucleotides were filter-sterilized and used at a concentration of 0.1-10 ⁇ M.
  • Example 3 Construction of Antisense Rat IGF IR Vector A diagrammatic representation of the vector assembly is shown in Figure 8. Briefly, the rat IGF IR cDNA clone pll ⁇ was digested by Xhol and Kpnl and the 0.8 kb restriction fragment (from nucleotide +277 to +1086 relative to ATG) was ligated in an antisense orientation into pCEP4 (Invitrogen) . (See also SEQ ID NO:8.) Transcription of the antisense IGF IR cDNA is under control of the cytomegalovirus immediate early gene enhancer/promoter, and yields a 1.0 kb antisense transcript
  • This vector contains two Epstein Barr virus (EBV) derived genes namely, the EBV ori-P (origin of replication) and the EBV-nuclear antigen 1, which allow episomal replication.
  • EBV ori-P epigen of replication
  • EBV-nuclear antigen 1 which allow episomal replication.
  • the SV40 poly(A) tail provides a termination signal for transcription, and a hygromycin resistance gene allows selection of transfectants.
  • vascular smooth muscle cells were isolated from rat thoracic aorta by enzymatic dissociation as described by Gunther et al. (1982) J. Cell . Biol . 21:289-298. They were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum (CS) , 2 mM glutamine, 100 units/ml penicillin and 100 ⁇ g/ml streptomycin, and passaged twice a week by harvesting with trypsin-versene and seeding at a 1:8 ratio in 75 cm 2 flasks.
  • CS calf serum
  • oligonucleotide experiments For oligonucleotide experiments, cells between passage levels 5 and 15 were seeded into 100-mm, 24-well or 48-well polystyrene cluster culture dishes. For certain experiments cells were quiesced by exposure to defined serum-free medium (SFM) containing DMEM and Ham's F12 (1:1) supplemented with transferrin (5 ⁇ g/ml), insulin (5 x 10 "7 M) , ascorbate (0.2 mM) , glutamine and antibiotics.
  • SFM serum-free medium
  • transferrin 5 ⁇ g/ml
  • insulin 5 x 10 "7 M
  • ascorbate 0.2 mM
  • glutamine glutamine
  • Example 5 Cell Transfection and Selection Vascular smooth muscle cells were isolated an cultured as described in Example 4 hereinabove. For transfections, -10% confluent (passage number five) cells in 100 mm dishes were incubated in the presence of 10 ⁇ g of calcium phosphate precipitate of carrier DNA (pRSV-CAT, Invitrogen) and the plasmid pAnti-IGF IR in a 1:2 ratio for six hours as described by Chen and Okayama* (1988) Bio Techniques 6.:632-638. Cells were grown in hygromycin-free medium for 72 hours and then selected in the presence of hygromycin (Sigma) at a concentration of 100 ⁇ g/ml. This concentration of antibiotic was determined to effectively kill VSMC. Selection medium was replaced every three days. After six weeks, hygromycin resistant colonies were isolated, cloned into 24 well plates and subsequently expanded into 12 and 6 well tissue culture plates.
  • pRSV-CAT calcium phosphate precipitate of carrier DNA
  • Example 6 Measurement of DNA synthesis
  • VSMC were grown to 80% confluence in 48-well plates, serum-deprived for 48 hr in the presence or absence of ODNs (0.1- 10 ⁇ M) and then exposed to fresh SFM in the absence of ODNs, or to 10% serum, for 24 hr.
  • ODNs 0.1- 10 ⁇ M
  • SFM serum-deprived for 48 hr
  • 1 ⁇ Ci/ml [ 3 H] -thymidine was included during the last 24 hr.
  • VSMC were grown to 50% confluence and then incubated in DMEM with 10% CS alone, or in the presence of 5 ⁇ M AS or 5 ⁇ M S ODN for 48 hr. Cells were then washed in serum-free medium (SFM) and incubated in SFM ⁇ IGF I (1-100 ng/ml) for 24 hr. 1 ⁇ Ci/ml [ 3 H] -thymidine was present during the latter 24 hr., and TCA-precipitable counts were determined as described above.
  • SFM serum-free medium
  • VSMC were exposed to AS or S ODNs (5 ⁇ M) for 48 hr in the presence of 10% CS. Cells were then washed in SFM and exposed to SFM ⁇ ang II (1-1000 nM) for 24 hr. At 24 hr cells were pulsed with [ 3 H] -thymidine (1 ⁇ Ci/ml) for 12 hr in the continued presence of ang II. TCA-precipitable counts were then determined as described above.
  • VSMC were grown to 50% confluence in 48-well plates and then exposed to SFM alone, 10% CS alone or 10% CS and increasing concentrations (0.1-10 ⁇ M) of AS, S, or M ODNs. Medium and ODNs were replaced at 48 hr. At 96 hr cells were trypsinized and counted. To analyze growth curves on cells with transcribed antisense IGF IR oligonucleotides, transfected cells were split into 24-well culture plates at a density of 2,000 cells per well, and grown in the presence of 10% calf serum and 100 ⁇ g/ml hygromycin. Cells in duplicate wells were washed with PBS, harvested with PBS/20 mM EDTA and counted manually. Culture medium replacement and cell counts were performed every two days. Example 8 Binding Assays
  • VSMC were grown to 80% confluence in 24- well plates, and then exposed to SFM alone or in the presence of AS, S, or M ODNs (5 ⁇ M) for 48 hr.
  • SFM serum-free medium
  • M ODNs 5 ⁇ M
  • cells were grown to 50% confluence and then exposed to 10% CS alone or in the presence of AS or S ODNs (1,5 ⁇ M) for 48 hr. Cells were washed in PBS and incubated at 37°C for 60 min.
  • binding buffer (20 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2 M MgS0 4 , 10 mM NaHC0 3 , 1.3 mM CaCl 2 , 1.2 mM KH 2 P0 4 , 0.25% bovine serum albumin, pH 7.4) to allow dissociation of cell-bound IGF I.
  • Cells were then rewashed and then incubated in the presence of 10 "10 M 125 I-IGF I and increasing (0-10 "7 M) concentrations of unlabeled IGF I for 90 min at room temperature. Cells were washed in ice-cold binding-buffer and solubilized in 2N NaOH before counting using an automated gamma counter with about 80% efficiency.
  • pGEM3 Promega Corp., Madison, WI
  • the subclone p26K was linearized with EcoRI to allow generation of antisense RNA probe using SP6 RNA polymerase.
  • Solution hybridization assays were performed by hybridizing 30 ⁇ g of total RNA with 5 x 10 5 cp [ 32 P] -UTP labelled AS IGF IR riboprobe and co-hybridizing with a glyceraldehyde-3- phosphate dehydrogenase (GAPDH) riboprobe as previously described (Delafontaine et al. (1991) Hypertension 19:693-699; Delafontaine et al. (1992) Hypertension 11:742-747; Delafontaine and Lou (1993) J. Biol . Chem. 286:16866-16870) .
  • GPDH glyceraldehyde-3- phosphate dehydrogenase
  • RNA was hybridized with 5 X 10 5 cpm of [ 3 P] -UTP labeled antisense IGF IR riboprobe and co-hybridized with a glyceraldehyde-3- phosphate dehydrogenase (GAPDH) riboprobe (Fort et al. (1985) Nucl . Acids Res . 13:1431-1442) i n a solution containing 80% deionized formamide, 40 mM PIPES, pH 6.4, 0.4 M NaCl, 1 mM EDTA.
  • GPDH glyceraldehyde-3- phosphate dehydrogenase
  • a 32 P-labeled rat IGF IR sense RNA probe (120 bp, nucleotides +560 to +680) was generated by T7 polymerase transcription of the rat IGR IR cDNA clone p522, after linearizing with PvuII.
  • Hybridization conditions were as recommended by Promega (Madison, WI) i.e., 7% PEG 8000, 7% SDS, 50% formamide, 0.25 M NaCl, 0.25 M NaP0 4 , pH 7.2, 100 ⁇ g/ml herring sperm DNA and 100 ⁇ g/ml yeast tRNA for 24 hr at 60°C. Filters were washed at room temperature in 2 x SSC and then at high stringency (0.1 x SSC, 30 min at 65°C) prior to autoradiography.
  • Example 11 Vectors for Local Synthesis of IGF IR-Regulating
  • a fragment of the IGF IR cDNA clone of at least about 100 bp and containing the region surrounding the ATG start site is isolated and purified for insertion in the vector.
  • These applications include stimulation of burn and other wound healing, angiogenesis and nerve regeneration.
  • the Xhol-Kpnl fragment of the IGF IR cDNA is used with insertion opposite in orientation to the expression control sequences (as in Example 3 herein) .
  • Other portions of the IGF IR cDNA of at least about 100 bp can be substituted.
  • Such applications include prevention of postangioplasty restenosis or atherosclerosis of coronary arteries following cardiac transplant.
  • Control vectors are those lacking an IGF IR-specific insert.
  • the vector used is a replication-deficient adenovirus vector
  • Example 12 Trans-Catheter Delivery of Oligonucleotides Domestic Buffalo pigs (12-15 kg) are used as an animal model in tests of antisense oligonucleotide therapy to prevent postangioplasty restenosis.
  • P gs are anesthetized with zolazepamin-tiletamine (6.0 mg/kg) in combination with intramuscular rompun (2.2 mg/kg) with 1% nitrous oxide.
  • the iliofemoral arteries are exposed using sterile surgical procedures, and a double-balloon catheter (C.R. Bard, Inc.) is inserted into the iliofemoral artery as previously described (Nabel et al. (1990) Science 249:1285; Nabel et al.
  • the proximal balloon is inflated to 500 mmHg, as measured using an on-line pressure transducer, for 5 min.
  • the balloon is then deflated and the catheter is advanced so that the central space between the proximal and distal balloons now 32 occupies the region of the previous injury. Both balloons are inflated, and the segment is irrigated with heparinized saline.
  • antisense oligonucleotide preparations comprising antisense oligonucleotide (SEQ ID NO:l) or mismatch oligonucleotide control (SEQ ID NO:3) 1 g per vessel, in saline, 2 ml volume, are instilled for 20 min in the central space in the catheter.
  • the catheter is removed, and antegrade blood flow is restored.
  • the arteries are analyzed 21 or 42 days later to determine vascular proliferation and/or restenosis.
  • measurements of intimal (I) and medial (M) areas are determined in at least four sections from each artery, and the measurements from each artery are averaged.
  • Comparisons of I/M area ratios for antisense-treated v. mismatch-treated are made using ANOVA with Dunnett's t test (Dunnet, C.W. (1984) Biometrics 2_0:482) .
  • Statistical significance is assumed if a null hypothesis could be rejected at the 0.05 level.
  • a recombinant DNA molecule capable of directing the expression of IGF IR-specific antisense RNA and preferably only transiently expressed is administered by the above procedure.
  • a recombinant molecule is described in Example 11 herein.
  • the adenovirus vector described therein is replication-deficient and therefore not stably maintained in the tissue.
  • An adenovirus vector can be used at an exemplary dose of about 5 x 10 9 to 1 x 10 10 plaque forming units, in a volume of about 0.7 ml.
  • Example 13 Materials Recombinant human IGF I was kindly provided by Dr. H.P. Guter, CIBA-GEIGY Corp. , Summit, NJ [ 3 H] -thymidine (20 Ci/mmole) , [ ⁇ - 32 P]UTP (3000 Ci/mmol) ] , [ ⁇ - 32 P]dCTP (3000 Ci/mmol) and 125 I-IGF I (-300 ⁇ Ci/ ⁇ g) , and [ ⁇ I-Sar ⁇ Ile 8 ] -ang II (2200 Ci/mmol) were obtained from Du Pont-New England Nuclear (Boston, MA) . Angiotensin II was purchased from Sigma Chemical Co. (St. Louis, MO) .
  • the coding sequence of the precursor protein extends from nucleotide 46 to nucleotide 4158, including the TGA stop codon within the encoded protein.
  • the signal peptide is from amino acids* -30 to -1 of SEQ ID NO:9.
  • the ⁇ -subunit begins at amino acid 1.
  • the C-terminus of the ⁇ -subunit is generated by protease cleavage at the 4-Arg proteolytic processing site (amino acids 708-711) .
  • the cysteine-rich region of the ⁇ -subunit is from amino acids 148 to 302.
  • the ⁇ -subunit extends from amino acids 712 to 1340 of SEQ ID NO: 9.
  • the ⁇ -subunit transmembrane domain extends from amino acids 908 to 924, and the tyrosine kinase domain within the ⁇ -subunit extends from amino acids 976 to 1084 Of SEQ ID NO:9.
  • the precursor nucleotide sequence and deduced amino acid sequence for human IGF I receptor extends from nucleotide 46 to 4149 of SEQ ID NO:10 (including the stop codon) .
  • the signal peptide extends from amino acids -30 to -1 of SEQ ID NO:11.
  • the mature ⁇ -subunit begins at amino acid 1 and extends to the proteolytic cleavage site at amino acids 707 to 710; the ⁇ - subunit begins at amino acid 711 and extends to amino acid 1337.
  • the ⁇ -subunit transmembrane domain is at amino acids 906 to 929.
  • MOLECULE TYPE other nucleic acid
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE other nucleic acid
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:5:
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FEATURE FEATURE:
  • CAG AAA ATG TGC CCA AGT GTG TGT GGG AAG CGA GCC TGC ACC GAG AAC 726 Gin Lys Met Cys Pro Ser Val Cys Gly Lys Arg Ala Cys Thr Glu Asn 185 190 195 AAT GAG TGC TGC CAC CCG GAG TGC CTA GGC AGC TGC CAC ACA CCG GAC 774 Asn Glu Cys Cys His Pro Glu Cys Leu Gly Ser Cys His Thr Pro Asp 200 205 210
  • CTTTTCTCCG CTTCCCAGTG GAAAGCCCAG CGGGGAAGCC TTTATTTATT TTTTTTAA 4608
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • GAG AAC AAG CTG CCC GAG CCG GAG GAG CTG GAC CTG GAG CCA GAG AAC 3942 Glu Asn Lys Leu Pro Glu Pro Glu Glu Leu Asp Leu Glu Pro Glu Asn 1255 1260 1265

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Abstract

Séquences de nucléotides et procédés permettant d'augmenter l'expression du récepteur du facteur de croissance de type I proche de l'insuline, en particulier pour favoriser la guérison de brûlures, de fractures osseuses et d'autres lésions, la régénération nerveuse dans les tissus lésés et l'angiogenèse. L'inhibition de l'angiogenèse et/ou de la croissance et de la prolifération cellulaires des muscles lisses de la paroi vasculaire peut être obtenue à l'aide d'ARN ou d'oligonucléotides antisens spécifiques de IGF IR. Des oligonucléotides sens dirigés vers ATG de IGF IR élèvent l'expression du gène IGF IR.
PCT/US1995/012563 1994-10-04 1995-09-27 Procedes de regulation du recepteur du facteur de croissance de type 1 proche de l'insuline WO1996010401A1 (fr)

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US5929040A (en) * 1994-07-08 1999-07-27 Royal Children's Hospital Research Foundation Method for the prophylaxis and/or treatment of proliferative and/or inflammatory skin disorders
US6468304B1 (en) 1997-07-16 2002-10-22 Centre National De La Recherche Scientifique Implantable device covered with polymer capable of releasing biologically active substances
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Cited By (12)

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US5929040A (en) * 1994-07-08 1999-07-27 Royal Children's Hospital Research Foundation Method for the prophylaxis and/or treatment of proliferative and/or inflammatory skin disorders
US6284741B1 (en) 1994-07-08 2001-09-04 Royal Children's Hospital Research Foundation Method for the prophylaxis and/or treatment of proliferative and /or inflammatory skin disorders
WO1998031381A1 (fr) * 1997-01-16 1998-07-23 University Of Florida Research Foundation, Incorporated Compostions permettant d'ameliorer les effets cytoprotecteurs de composes polycycliques phenoliques par interaction synergique avec des antioxydants
US5972923A (en) * 1997-01-16 1999-10-26 University Of Florida Research Foundation, Inc. Methods and compositions to enhance the cytoprotective effects of polycyclic phenolic compounds through the synergistic interaction with anti-oxidants
AU719764B2 (en) * 1997-01-16 2000-05-18 University Of Florida Research Foundation, Inc. Compositions to enhance the cytoprotective effects of polycyclic phenolic compounds through the synergistic interaction with anti-oxidants
US6468304B1 (en) 1997-07-16 2002-10-22 Centre National De La Recherche Scientifique Implantable device covered with polymer capable of releasing biologically active substances
US6900186B1 (en) 1999-06-21 2005-05-31 Murdock Children's Research Institute Method for the prophylaxis and/or treatment of medical disorders
WO2004072284A1 (fr) * 2003-02-11 2004-08-26 Antisense Therapeutics Ltd Modulation de l'expression du recepteur du facteur de croissance i analogue a l'insuline
JP2006520586A (ja) * 2003-02-11 2006-09-14 アンチセンス セラピューティクス リミテッド インスリン様増殖因子ｉ受容体発現の調節
US7468356B2 (en) 2003-02-11 2008-12-23 Antisense Therapeutics Ltd. Modulation of insulin like growth factor I receptor expression
JP4753863B2 (ja) * 2003-02-11 2011-08-24 アンチセンス セラピューティクス リミテッド インスリン様増殖因子ｉ受容体発現の調節
US8217017B2 (en) 2003-02-11 2012-07-10 Antisense Therapeutics Limited Modulation of insulin like growth factor I receptor expression

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