WO2012170250A1 - Morpholino oligonucleotides capable of inhibiting cd47-mediated cellular damage and uses thereof - Google Patents

Abstract

The present invention relates to morpholino oligonucleotides that exhibit high-affinity and high specificity for binding to CD47, and which have the ability to inhibit the translation of CD47. The invention further relates to the use of such morpholino oligonucleotides in radioprotection, and in the treatment of cancer, heart disease, CNS disorders and inflammation.

Description

Title of the Invention:

Morpholino Oligonucleotides Capable of Inhibiting CD47-Mediated Cellular Damage and Uses Thereof

Cross-Reference to Related Applications:

[0001] This application claims priority to United States Patent Application No. 61/494,016 (filed June 7, 2011; pending), which application is herein incorporated by reference in its entirety.

Reference to Sequence Listing:

[0002] This application includes one or more Sequence Listings pursuant to 37 C.F.R. 1.821 et seq., which are disclosed in both paper and computer-readable media, and which paper and computer-readable disclosures are herein incorporated by reference in their entirety.

Background of the Invention:

Field of the Invention:

[0003] The present invention relates to morpholino oligonucleotides that exhibit high-affinity and high specificity for binding to CD47, and which have the ability to inhibit the translational synthesis of CD47 and its interaction with thrombospondin 1. The invention further relates to the use of such morpholino oligonucleotides in radioprotection, and in the treatment of cancer, heart disease, CNS disorders and inflammation.

Description of Related Art:

I. Radiation-Induced Damage

Ionizing radiation has been used in both diagnostic and therapeutic medical applications (Baskar, R. (2010) "Emerging Role Of Radiation Induced Bystander Effects: Cell Communications And Carcinogenesis," Genome Integrity content 1/1/13). Such use, however, raised considerable concerns about the detrimental health effects associated with direct radiation exposure (Hall, E.J. (2000) "Radiation, The Two-Edged Sword: Cancer Risks At High And Low Doses," Cancer J. 6:343-350; Little, J.B. (2003) "Genomic Instability And Bystander Effects: A Historical Perspective," Oncogene 22:6978-6987) even on metabolically inactive cells.

[0004] Radiotherapy continues to be an important therapeutic modality for the treatment of cancer even though its use reveals a risk of adverse consequences, such as tissue atrophy and formation of secondary tumors at the same organ, or at some distanced part of body (Widener, A. (2006) "Radiation increases risk of second primary tumors for childhood survivors," J. Natl. Cancer Inst. 98: 1507). Additionally, radiotherapy is increasingly being employed in routine diagnostics (e.g. , X-rays, CT-scans, etc. and thus affecting the health of the general population.

[0005] Such increased use of radiotherapy (particularly in children who are inherently more radiosensitive than adults), coupled with the enhanced survival of cancer patients, increases the possibility of radiation-induced secondary cancers in patients treated with ionizing radiation (Baskar, R. (2010) "Emerging Role Of Radiation Induced Bystander Effects: Cell Communications And Carcinogenesis," Genome Integrity content 1/1/13).

[0006] Damage of DNA is central to major biological processes such as cancer, disturbances of cell function and aging. In normal cells, DNA damage of a single cell most likely represents an initiating event for carcinogenesis. The DNA damage response system, which maintains the survival and genomic stability of the cell, represents a vital line of defense against various exogenous and endogenous DNA damaging agents (Madhusudan, S. et al. (2005) "The Emerging Role Of DNA Repair Proteins As Predictive, Prognostic And Therapeutic Targets In Cancer," Cancer Treat. Rev. 31 :603-607). Radiation can induce apoptosis or trigger DNA repair mechanisms. In general minor DNA damage is thought to halt cell cycle transit in order to allow effective repair, while more severe damage can induce an apoptotic cell death program.

[0007] Recent observations have challenged the conclusion that genetic and biochemical alterations are restricted solely to those cells that experienced direct irradiation, since similar damage effects can also be seen in normal, non-irradiated cells adjacent to the irradiated or targeted cells (the "bystander effect") (Blyth, B.J. et al. (June 2011) "Radiation-Induced Bystander Effects: What Are They, and How Relevant Are They to Human Radiation Exposures? '," Radiat. Res. (doi: 10.1667/RR2548.1); Fischer, P. et al. (May 2011) "The Bystander-Effect: A Meta- Analytic Review On Bystander Intervention In Dangerous And Non-Dangerous Emergencies;' Psychol. Bull, (doi: 10.1037/a0023304); Baskar, R. (2010) "Emerging Role Of Radiation Induced Bystander Effects: Cell Communications And Carcinogenesis " Genome Integr. 1(1): 13 (1-8); Burdak-Rothkamm, S. et al. (2009) "New Molecular Targets In Radiotherapy: DNA Damage Signalling And Repair In Targeted And Non-Targeted Cells " Eur. J. Pharmacol. 625(1-3): 151-155; Rzeszowska-Wolny, J. et al. (2009) "Ionizing Radiation-Induced Bystander Effects, Potential Targets For Modulation Of Radiotherapy " Eur. J. Pharmacol. 625(1- 3):156-64; Morgan, W.F. et al. (2009) "Non-Targeted Effects Of Ionizing Radiation: Implications For Risk Assessment And The Radiation Dose Response Profile " Health Phys. 97(5):426-432; Munro, A.J. (2009) "Bystander Effects And Their Implications For Clinical Radiotherapy " J. Radiol. Prot. 29(2A):A133-A142). In light of the "bystander effect" it is increasingly being seen as important to provide radioprotective treatment to normal cells of patients being subjected to radiotherapy. Thus, researchers have attempted to understand the molecular basis of the "bystander effect."

II. Thrombospondin-1 and CD47

[0008] The thrombospondins comprise a family of five genes encoding proteins designated TSP-1 through TSP-5 (Adams, J.C. et al. (1995) "The Thrombospondin Gene Family " R.G. Landes Company, Austin, TX). Thrombospondin-1 (TSP1) is a large homotrimeric glycoprotein that is synthesized and secreted by many cell types in response to injury and certain growth factors (Isenberg, J.S. et al. (2008) "Thrombospondin-1 is a Central Regulator of Nitric Oxide Signaling in Vascular Physiology " Cell. Mol. Life Sci. 65(5):728-742). Although thrombospondin-1 is expressed at various sites during development and is present at low levels in normal plasma, high level expression in healthy adults is largely restricted to megakaryocytes, circulating platelets, and sites of tissue remodeling or injury (Isenberg, J.S. et al. (2008) "Thrombospondin- 1 is a Central Regulator of Nitric Oxide Signaling in Vascular Physiology," Cell. Mol. Life Sci. 65(5):728-742).

[0009] Thrombospondin-1 has been found to interact with at least 9 cell receptors on endothelial cells including α3β1, α4β1, α6β1, α9β1, and ανβ3 integrins, the LDL receptor-related protein (LRP) in association with calreticulin, heparan sulfate proteoglycans, CD36, and CD47 (Isenberg, J.S. et al. (2008) " ^'Thrombospondin-1 is a Central Regulator of Nitric Oxide Signaling in Vascular Physiology " Cell. Mol. Life Sci. 65(5):728-742).

[0010] The interaction of thrombospondin-1 with CD47 is of particular significance to the present invention. CD47 is a member of the immunoglobulin (Ig) superfamily of membrane proteins, with a single IgV-like domain at its N-terminus, a highly hydrophobic stretch with five membrane-spanning segments and an alternatively spliced cytoplasmic C-terminus ranging in length from 3-36 amino acids (Brown, E.J. et al. (2001) "Integrin- Associated Protein (CD47) And Its Ligands " Trends Cell Biol. 11(3): 130-135; Gao A.G. et al. (1996) "Integrin- Associated Protein Is A Receptor For The C-Terminal Domain Of Thrombospondin," J. Biol. Chem. 271 :21-24; Wang, X.Q. et al. (1998) "The Thrombospondin Receptor CD47 (IAP) Modulates And Associates With Alpha2 Betal Integrin In Vascular Smooth Muscle Cells " Mol. Biol. Cell. 9(4):865-874). The C-terminal domain of thrombospondin-1 interacts with the Ig domain of CD47 to activate CD47. Such activation modulates the activity of several integrins (Gao A.G. et al. (1996) Integrin- Associated Protein Is A Receptor For The C-Terminal Domain Of Thrombospondin " J. Biol. Chem. 271 :21-24.

[0011] In particular, the activation of thrombospondin has been found to inhibit the body's response to nitric oxide (United States Patent Publication No. 2010/0092467, herein incorporated by reference). Nitric oxide has been found to mediate vasodilation and increased blood flow, and to thereby serve as a means for protecting cells from damage and death (Gladwin, M.T. et al. (2006) 'Nitrite As A Vascular Endocrine Nitric Oxide Reservoir That Contributes To Hypoxic Signaling, Cytoprotection, And Vasodilation " Am. J. Physiol. Heart Circ. Physiol. 291 :H2026- H2035) [0012] Under hypoxic conditions such as during ischemia or myocardial infarction, nitric oxide (NO) is produced from the conversion of L-arginine to L-citrulline, through the nitrite reductase activities of hemoglobin, myoglobin, xanthine oxidase and other proteins, or by other means (Isenberg, J.S. et al. (2008) "Thrombospondin-I is a Central Regulator of Nitric Oxide Signaling in Vascular Physiology,^'" Cell. Mol. Life Sci. 65(5):728-742; Gladwin, M.T. et al. (2006) 'Nitrite As A Vascular Endocrine Nitric Oxide Reservoir That Contributes To Hypoxic Signaling, Cytoprotection, And Vasodilation " Am. J. Physiol. Heart Circ. Physiol. 291 :H2026- H2035; Shiva, S. et al. (2007) "Deoxymyoglobin Is A Nitrite Reductase That Generates Nitric Oxide And Regulates Mitochondrial Respiration,"Circ. Res. 100:654-661; Rassaf, T. et al. (2007) "Nitrite Reductase Function Of Deoxymyoglobin: Oxygen Sensor And Regulator Of Cardiac Energetics And Function " J. Circ. Res. 100: 1749-1754)). The produced nitric oxide acts to promote the relaxation of vascular smooth muscle cells and thus increases blood vessel diameter and blood flow to effect the reversal of the hypoxic state.

[0013] Because thrombospondin-1, through its interaction with CD47, inhibits nitric oxide signaling, it thus serves to impair the normal response to ischemia and to promote apoptosis and cell death (Isenberg, J.S. et al. (2006) "CD47 Is Necessary For Inhibition Of Nitric Oxide-Stimulated Vascular Cell Responses By Thrombospondin- 1," J. Biol. Chem. 281 :26069-26080; Kaczorowski, D.J. et al. (2011) "Targeting CD47 - NO Limit on Therapeutic Potential " Circ. Res. 100;602-603) promotes cell death (Isenberg, J.S. et al. (2008) "Thrombospondin-1 is a Central Regulator of Nitric Oxide Signaling in Vascular Physiology " Cell. Mol. Life Sci. 65(5):728-742).

[0014] Agents that disrupt such inhibition have been proposed as potential cell protectors, and as radioprotective agents.

III. Morpholino Oligonucleotides

[0015] Antisense molecules have been investigated as potential blocking agents of transcribed genes. Antisense molecules are molecules that are specifically hybridizable or specifically complementary to either RNA or "plus" strand DNA. Such molecules thus have the potential for specifically hybridizing to a target mRNA and inhibiting its transcription. Antisense agents are highly selective inhibitors or modulators of gene expression and range from plasmid vectors that express antisense R A molecules several nucleotides long to short single-stranded oligonucleotides consisting of 10-50 nucleotide residues. Their ability to inhibit or modulate gene expression in a sequence-specific manner offers the potential for high specificity in immediate therapeutic applications. Several preclinical and clinical trials are currently ongoing to evaluate the use of antisense oligonucleotides as therapeutic agents (Amantana, A. et al. (2005) "Pharmacokinetics And Biodistribution Of Phosphorodiamidate Morpholino Antisense Oligomers" Current Opinion in Pharmacology 5:550-555). One deficiency of antisense molecules, however, is the susceptibility of the produced R A/DNA hybrid to cleavage by RNAse H.

[0016] Morpholino oligonucleotides have been proposed as a means for surmounting this obstacle. Morpholino oligonucleotides are structurally different from natural nucleic acids. They possess morpholino rings in lieu of the ribose or deoxyribose sugar moieties of RNA or DNA, and possess non-ionic phosphorodiamidate linkages in lieu of the anionic phosphates of RNA or DNA. Each morpholino ring suitably positions one of the standard bases (A, G, C, T/U), so that a 25 -base morpholino oligonucleotide strongly and specifically binds to its complementary 25-base target site in a strand of RNA via Watson-Crick pairing. However, because the backbone of the morpholino oligonucleotide is not recognized by cellular enzymes of signaling proteins, it is stable to nucleases and does not trigger an innate immune response through the toll-like receptors. This avoids loss of the oligonucleotide, as well as attenuates inflammation or interferon induction. Morpho linos can be delivered by a number of techniques, including direct injection to tissues or via infusion pump and intravenous bolus (United States Patent Publication No. 2010/0092467). Morpholino oligonucleotides have no electrical charge, and do not interact strongly with proteins, and do not require the activity of RNase-H, Argonaute, or other catalytic proteins for their activity (Moulton, J.D. et al. (2009) "Gene Knockdowns in Adult Animals: PPMOs and Vivo-Morpholinos," Molecules 14: 1304-1323). [0017] Morpholino oligonucleotides are well-known in the art (see, e.g., Amantana,

A. et al. (2005) "Pharmacokinetics And Biodistribution Of Phosphorodiamidate Morpholino Antisense Oligomers" Current Opinion in Pharmacology 5:550-555; Bill,

B. R. et al. (2009) "A Primer for Morpholino Use in Zebrafish " Zebrafish 6(l):69-77; Heasman, J. (2002) "Morpholino Oligos: Making Sense of Antisense? ^'," Devel. Biol. 243:209-214; Karkare, S. et al. (2006) "Promising Nucleic Acid Analogs And Mimics: Characteristic Features And Applications Of PNA, LNA, And Morpholino," Appl. Microbiol. Biotechnol. 71 :575-586; Moulton, J.D. et al. (2009) "Gene Knockdowns in Adult Animals: PPMOs and Vivo-Morpholinos," Molecules 14: 1304-1323; Moulton, J.D. et al. (2010) "Morpholinos And Their Peptide Conjugates: Therapeutic Promise And Challenge For Duchenne Muscular Dystrophy," Biochimica et Biophysica Acta 1798:2296-2303).

[0018] In particular, Isenberg, J.S. et al. (2008) ("Thrombospondin-1 is a Central Regulator of Nitric Oxide Signaling in Vascular Physiology," Cell. Mol. Life Sci. 65(5):728-742) suggested using an antisense morpholino oligonucleotide complementary the 5' region of murine and human CD47 mRNA to prevent translation of this mRNA in vascular cells in vitro (Isenberg, J.S. et al. (2007) "Increasing Survival of Ischemic Tissue by Targeting CD47," Circ. Res. 100:712- 720). The morpholino was also effective in FTSG (Isenberg, J.S. et al. (2008) "Blockade Of Thrombospondin-1 -CD47 Interactions Prevents Necrosis Of Full Thickness Skin Grafts," Ann. Surg. 247(1): 180-190); see also, United States Patent Publication No. 2010/0092467).

[0019] Despite such advances, a need remains for improved morpholino oligonucleotides capable of exhibiting tighter binding or greater specificity to CD47. The present application is directed to such improved compounds and their uses.

Summary of the Invention:

[0020] The present invention relates to morpholino oligonucleotides that exhibit high-affinity and high specificity for binding to CD47, and which have the ability to inhibit the translational synthesis of CD47 and its interaction with thrombospondin 1. The invention further relates to the use of such morpholino oligonucleotides in radioprotection, and in the treatment of cancer, heart disease, CNS disorders and inflammation.

[0021] In detail, the invention provides a morpholino oligonucleotide agent that inhibits interaction between thrombospondin-1 (TSP1) and CD47 and/or the translational synthesis of CD47, as well as the use of that agent in a method of protecting animal tissue from damage caused by radiation exposure, comprising contacting the tissue with a therapeutically effective amount of the agent.

[0022] The invention additionally provides such an agent and use, wherein the agent is employed in a method of protecting a subject exposed (or at risk of exposure) to a radioactive substance or ionizing radiation, comprising contacting tissue of the subject with the therapeutically effective amount of the agent.

[0023] The invention additionally provides such an agent and use, wherein the contacting is performed at least one of before, during or after exposure to radiation, and preferably, wherein the agent is administered within two weeks prior to exposure to radiation, during radiation exposure, and/or within two weeks following radiation exposure, or wherein the agent is administered within four days prior to radiation exposure, during radiation exposure, and/or within about one day following radiation exposure.

[0024] The invention additionally provides such an agent and use, wherein the radiation comprises an acute or chronic dose of ionizing or non-ionizing radiation and preferably, wherein the ionizing radiation (i) results from nuclear fission or fusion, or (ii) comprises X-rays, or (iii) comprises radionuclides.

[0025] The invention additionally provides such an agent and use, wherein the agent is employed in a method of enhancing the therapeutic window for radiotherapy in a subject, comprising contacting tissue of the subject with the therapeutically effective amount of the agent prior to the radiotherapy.

[0026] The invention additionally provides such an agent and use, wherein the radiation exposure comprises diagnostic X-rays, radiation therapy, a CAT-scan, a mammogram, a radionuclide scan, or an interventional radiological procedure under CT or fluoroscopy guidance.

[0027] The invention additionally provides such an agent and use, wherein the radiation exposure comprises tissue -incorporated radionuclides from ingestion of contaminated food or water, non-medical or unintentional exposure to ionizing radiation.

[0028] The invention additionally provides an agent that inhibits interaction between TSP1 and CD47 for use in a method of increasing tumor ablation in a subject undergoing radiotherapy, comprising contacting tissue of the subject with a therapeutically effective amount of the agent, which agent comprises: an oligonucleotide comprising at least about 15 contiguous bases and that hybridizes to the mRNA of CD47 with 0, 1, 2, 3, 4, or 5 mismatches.

[0029] The invention additionally provides such an agent wherein the agent is administered topically, buccally, intraocularly, orally, subcutaneously, intramuscularly, intravenously, intraarterially, transdermally, intranasally, rectally, peritoneally, or by inhalation.

[0030] The invention additionally provides such an agent for use in any of the above-described methods, wherein inhibiting interaction between TSP1 and CD47 comprises one or more of the following:

(a) inhibiting expression of CD47;

(b) removing endogenous CD47;

(c) blockading interaction between endogenous TSP1 and CD47.

[0031] The invention additionally provides such an agent wherein (i) the agent comprises an oligonucleotide morpholino comprising at least about 15 contiguous bases and that hybridizes to the mRNA of CD47 under high stringency conditions, and preferably, wherein the oligonucleotide is a morpholino and preferably, wherein the morpholino comprises the sequence shown in SEQ ID NO: 6, 11, 3 or 9.

[0032] The invention additionally provides such an agent to render cancer sensitive to radiation killing by any form of therapeutically applied form of radiation. [0033] The invention additionally provides such an agent that specifically is employed in the course of radiation therapy that render soft tissue carcinoma more sensitive to radiation such that lower total radiation is required to achieve cancer killing.

[0034] The invention additionally provides such an agent that specifically is employed to render blood borne cancers more susceptible to radiation killing.

[0035] The invention additionally provides such an agent that inhibits the interaction of thrombospondin-1 (TSP-1) and CD47 for use in a method of increasing tissue perfusion in a subject, comprising selecting a subject in need of increased tissue perfusion; and administering to the subject a therapeutically effective amount of the agent, thereby increasing tissue perfusion in the subject.

[0036] The invention additionally provides such an agent that inhibits function or expression of CD47 for use in method comprising selective application to a subject of the one or more agents, which method: improves tissue or organ survival in the subject; treats or ameliorates peripheral vascular disease or myocardial ischemia in the subject; improves blood flow in the subject;

(a) improves transplant organ or tissue survival in the subject;

(b) improves wound healing of acute and/or chronic wounds;

(c) improves healing of surgical wounds;

(d) improves healing of traumatic injuries;

(e) improves burn survival or recover in the subject;

(f) improves skin graft survive in the subject; and/or

(g) improves survival of reattached extremities and body parts.

[0037] The invention additionally provides an agent that influences interaction between TSP-1 and CD47 for use in a method of controlling blood pressure in a subject.

[0038] The invention additionally concerns the above agents and uses wherein the subject has diabetes. [0039] The invention additionally provides an agent that inhibits the interaction of thrombospondin-1 (TSP-1) and CD47 for use in a method of treating tissue necrosis resulting from ischemia in an elderly subject with atherosclerotic vascular disease or age-related vasculopathy, comprising: selecting an elderly subject with necrosis in a tissue, wherein the subject has atherosclerotic disease or age-related vasculopathy; and administering to the subject a therapeutically effective amount of the agent, thereby treating the tissue necrosis.

[0040] The invention also concerns the above-described agents, wherein the elderly subject: has atherosclerosis, has a graft; has had a myocardial infarction; has a vasculopathy, has Alzheimer's disease; has dementia; or any combination of two or more thereof.

Detailed Description of the Invention:

[0041] The present invention relates to morpholino oligonucleotides that exhibit high-affinity and high specificity for binding to CD47, and which have the ability to inhibit the translation of CD47. The novel compositions disclosed herein are third generation antisense oligomer therapeutics, specifically 15-25 nucleic acid phosphorodiamidate morpholino oligomers (PMO).

[0042] PMO's behave like small molecule therapeutics and have an outstanding safety record in multiple clinical trials. PMO's are highly specific, stable, effective and nontoxic (Shrewsbury, S.B. (2010) "Preclinical Safety Of A VI-4658, A Phosphorodiamidate Morpholino Oligomer (PMO), Being Developed To Skip Exon 51 In Duchenne Muscular Dystrophy (DMD)," AVI Biopharma, Inc.; Press Release (2011) "Systemic Treatment With AVI-4658 Demonstrates RNA Exon Skipping and Dystrophin Protein Expression in Duchenne Muscular Dystrophy Patients," AVI Biopharma, Inc.). They generally comprise about 20-25 nucleic acid bases linked by an uncharged synthetic backbone. They bind to complementary sequences of RNA by base pairing to prevent processes from happening at the bound sites. They may be targeted to stop the progression of a ribosomal initiation complex toward the start codon of an mRNA, preventing protein translation. They may also be targeted to interfere with pre -mRNA splicing machinery, altering content of mature mRNA. [0043] The coding sequence of murine CD47 is provided in NM 010581.3; the sequence of human CD47 (transcript variant 1) is provided in NM_001777.3.

[0044] NM 010581.3 (SEQ ID NO: 1) (the murine CD47 encoding sequence is shown underlined):

cccgggcagc ctgggcggcc gctcctgcct gtcactgctg cggcgctgct ggtcggtcgt ttcccttgaa ggcagcagcg gaggcggcgg ctgctccaga cacctgcggc ggcgaccccc cggcggcgcg gagatgtggc ccttggcggc ggcgctgttg ctgggctcct gctgctgcgg ttcagctcaa ctactgttta gtaacgtcaa ctccatagag ttcacttcat gcaatgaaac tgtggtcatc ccttgca cg tccgtaatgt ggaggcgcaa agcaccgaag aaatgtttgt gaagtggaag ttgaacaaat cgtatatttt catctatgat ggaaataaaa a agcactac tacagatcaa aactttacca gtgcaaaaat ctcagtc ca gacttaa ca a ggcattgc ctctttgaaa a ggataagc gcgatgccat ggtgggaaac tacacttgcg aagtgacaga gttatccaga gaaggcaaaa cagttataga gctgaaaaac cgcacggcct tcaacactga ccaagga ca gcctgttctt acgaggagga gaaaggaggt tgcaaattag tttcgtggtt ttctccaaat gaaaagatcc tcattg at tttcccaatt ttggctatac tcc gttctg gggaaagttt ggtattttaa cactcaaata taaatccagc catacgaata agagaa cat tctgctgctc gttgccgggc tggtgctcac agtcatcgtg gttgttggag ccatccttct catcccagga gaaaagcccg tgaagaatgc ttctggactt ggcctcattg taatctctac ggggatatta a actacttc agtacaatgt gtttatgaca gcttttggaa tgacctcttt caccattgcc a attgatca ctcaagtgct gggctacgtc cttgctttgg tcgggctgtg tctctgcatc a ggcatgtg agccagtgca cggccccctt ttgatttcag gtttggggat catagctcta gcagaactac ttggattagt ttatatgaag tttgtcgctt ccaaccagag gactatccaa cctcctagga ataggtgaag ggaagtgacg gactgtaact tggaagtcag aaatggaaga atacagttgt ctaagcacca ggtcttcacg actcacagct ggaaggaaca gacaacagta actgacttcc atccaggaaa acatgtcaca taaatgatta ctaagtttat attcaaagca gctgtacttt acataataaa aaaaatatga tgtgctgtgt aaccaattgg aatcccattt ttctattgtt tctactcaac taggggcaaa cgtttcaggg gcaacttcca agaatgatgc ttgttagatc ctagagtctc tgaacactga gtttaaattg attccgagtg agactcgcca agcactaacc tgagggttag ttacccagag atacctatga aaaacagtgg tatccagcaa gccttagtaa actcaggttg ccagcagctt tgccacttcc gctgctagct gaataacaag actgccactt ctgggtcata gtgatagaga ctgaagtaga aaaacgaatg tggttgggca aatcccgtgt ggcccctctg tgtgctatga tattgatggc actggtgtct tcattcttgg gggttgccat cattcacaca cacccctttg acatacagtg caccccagtt ttgaatacat tttttttgca ccctgtcccg ttctgctact ttgatttgcg ttatgatata tatatatata tataatacct tttctcctct ttaaacatgg tcctgtgaca caatagtcag ttgcagaaag gagccagact tattcgcaaa gcactgtgct caaactcttc agaaaaaaaa aaaaaaaa

[0045] NM_001777.3 (SEQ ID NO: 2) (the human CD47 encoding sequence is shown underlined):

ggggagcagg cgggggagcg ggcgggaagc agtgggagcg cgcgtgcgcg cggccgtgca gcctgggcag tgggtcctgc ctgtgacgcg cggcggcggt cggtcctgcc tgtaacggcg gcggcggctg ctgctccaga cacctgcggc ggcggcggcg accccgcggc gggcgcggag atgtggcccc tggtagcggc gctgttgctg ggctcggcgt gctgcggatc agctcagcta ctatttaata aaacaaaatc tgtagaattc acgttttgta atgacactgt cgtcattcca tgctttgtta ctaatatgga ggcacaaaac actactgaag tatacgtaaa gtggaaattt aaaggaagag atatttacac ctttgatgga gctctaaaca agtccactgt ccccactgac tttagtagtg caaaaattga agtctcacaa ttactaaaag gagatgcctc tttgaagatg gataagagtg atgctgtctc acacacagga aactacactt gtgaagtaac agaattaacc agagaaggtg aaacgatcat cgagctaaaa tatcgtgttg tttcatggtt ttctccaaat gaaaatattc ttattgttat tttcccaatt tttgctatac tcctgttctg gggacagttt ggtattaaaa cacttaaata tagatccggt ggtatggatg agaaaacaat tgctttactt gttgctggac tagtgatcac tgtcattgtc attgttggag ccattctttt cgtcccaggt gaatattcat taaagaatgc tactggcctt ggtttaattg tgacttctac agggatatta atattacttc actactatgt gtttagtaca gcgattggat taacctcctt cgtcattgcc atattggtta ttcaggtgat agcctatatc ctcgctgtgg ttggactgag tctctgtatt gcggcgtgta taccaatgca tggccctctt ctgatttcag gtttgagtat cttagctcta gcacaattac ttggactagt ttatatgaaa tttgtggctt ccaatcagaa gactatacaa cctcctagga aagctgtaga ggaacccctt aatgcattca aagaatcaaa aggaatgatg aatgatgaat aactgaagtg aagtgatgga ctccgatttg gagagtagta agacgtgaaa ggaatacact tgtgtttaag caccatggcc ttgatgattc actgttgggg agaagaaaca agaaaagtaa ctggttgtca cctatgagac ccttacgtga ttgttagtta agtttttatt caaagcagct gtaatttagt taataaaata attatgatct atgttgtttg cccaattgag atccagtttt ttgttgttat ttttaatcaa ttaggggcaa tagtagaatg gacaatttcc aagaatgatg cctttcaggt cctagggcct ctggcctcta ggtaaccagt ttaaattggt tcagggtgat aactacttag cactgccctg gtgattaccc agagatatct atgaaaacca gtggcttcca tcaaaccttt gccaactcag gttcacagca gctttgggca gttatggcag tatggcatta gctgagaggt gtctgccact tctgggtcaa tggaataata aattaagtac aggcaggaat ttggttggga gcatcttgta tgatctccgt atgatgtgat attgatggag atagtggtcc tcattcttgg gggttgccat tcccacattc ccccttcaac aaacagtgta acaggtcctt cccagattta gggtactttt attgatggat atgttttcct tttattcaca taaccccttg aaaccctgtc ttgtcctcct gttacttgct tctgctgtac aagatgtagc accttttctc ctctttgaac atggtctagt gacacggtag caccagttgc aggaaggagc cagacttgtt ctcagagcac tgtgttcaca cttttcagca aaaatagcta tggttgtaac atatgtattc ccttcctctg atttgaaggc aaaaatctac agtgtttctt cacttctttt ctgatctggg gcatgaaaaa agcaagattg aaatttgaac tatgagtctc ctgcatggca acaaaatgtg tgtcaccatc aggccaacag gccagccctt gaatggggat ttattactgt tgtatctatg ttgcatgata aacattcatc accttcctcc tgtagtcctg cctcgtactc cccttcccct atgattgaaa agtaaacaaa acccacattt cctatcctgg ttagaagaaa attaatgttc tgacagttgt gatcgcctgg agtactttta gacttttagc attcgttttt tacctgtttg tggatgtgtg tttgtatgtg catacgtatg agataggcac atgcatcttc tgtatggaca aaggtggggt acctacagga gagcaaaggt taattttgtg cttttagtaa aaacatttaa atacaaagtt ctttattggg tggaattata tttgatgcaa atatttgatc acttaaaact tttaaaactt ctaggtaatt tgccacgctt tttgactgct caccaatacc ctgtaaaaat acgtaattct tcctgtttgt gtaataagat attcatattt gtagttgcat taataatagt tatttcttag tccatcagat gttcccgtgt gcctctttta tgccaaattg attgtcatat ttcatgttgg gaccaagtag tttgcccatg gcaaacctaa atttatgacc tgctgaggcc tctcagaaaa ctgagcatac tagcaagaca gctcttcttg aaaaaaaaaa tatgtataca caaatatata cgtatatcta tatatacgta tgtatataca cacatgtata ttcttccttg attgtgtagc tgtccaaaat aataacatat atagagggag ctgtattcct ttatacaaat ctgatggctc ctgcagcact ttttccttct gaaaatattt acattttgct aacctagttt gttactttaa aaatcagttt tgatgaaagg agggaaaagc agatggactt gaaaaagatc caagctccta ttagaaaagg tatgaaaatc tttatagtaa aattttttat aaactaaagt tgtacctttt aatatgtagt aaactctcat ttatttgggg ttcgctcttg gatctcatcc atccattgtg ttctctttaa tgctgcctgc cttttgaggc attcactgcc ctagacaatg ccaccagaga tagtggggga aatgccagat gaaaccaact cttgctctca ctagttgtca gcttctctgg ataagtgacc acagaagcag gagtcctcct gcttgggcat cattgggcca gttccttctc tttaaatcag atttgtaatg gctcccaaat tccatcacat cacatttaaa ttgcagacag tgttttgcac atcatgtatc tgttttgtcc cataatatgc tttttactcc ctgatcccag tttctgctgt tgactcttcc attcagtttt atttattgtg tgttctcaca gtgacaccat ttgtcctttt ctgcaacaac ctttccagct acttttgcca aattctattt gtcttctcct tcaaaacatt ctcctttgca gttcctcttc atctgtgtag ctgctctttt gtctcttaac ttaccattcc tatagtactt tatgcatctc tgcttagttc tattagtttt ttggccttgc tcttctcctt gattttaaaa ttccttctat agctagagct tttctttctt tcattctctc ttcctgcagt gttttgcata catcagaagc taggtacata agttaaatga ttgagagttg gctgtattta gatttatcac tttttaatag ggtgagcttg agagttttct ttctttctgt tttttttttt tgtttttttt tttttttttt tttttttttt ttttgactaa tttcacatgc tctaaaaacc ttcaaaggtg attatttttc tcctggaaac tccaggtcca ttctgtttaa atccctaaga atgtcagaat taaaataaca gggctatccc gtaattggaa atatttcttt tttcaggatg ctatagtcaa tttagtaagt gaccaccaaa ttgttatttg cactaacaaa gctcaaaaca cgataagttt actcctccat ctcagtaata aaaattaagc tgtaatcaac cttctaggtt tctcttgtct taaaatgggt attcaaaaat ggggatctgt ggtgtatgta tggaaacaca tactccttaa tttacctgtt gttggaaact ggagaaatga ttgtcgggca accgtttatt ttttattgta ttttatttgg ttgagggatt tttttataaa cagttttact tgtgtcatat tttaaaatta ctaactgcca tcacctgctg gggtcctttg ttaggtcatt ttcagtgact aatagggata atccaggtaa ctttgaagag atgagcagtg agtgaccagg cagtttttct gcctttagct ttgacagttc ttaattaaga tcattgaaga ccagctttct cataaatttc tctttttgaa aaaaagaaag catttgtact aagctcctct gtaagacaac atcttaaatc ttaaaagtgt tgttatcatg actggtgaga gaagaaaaca ttttgttttt attaaatgga gcattattta caaaaagcca ttgttgagaa ttagatccca catcgtataa atatctatta accattctaa ataaagagaa ctccagtgtt gctatgtgca agatcctctc ttggagcttt tttgcatagc aattaaaggt gtgctatttg tcagtagcca tttttttgca gtgatttgaa gaccaaagtt gttttacagc tgtgttaccg ttaaaggttt ttttttttat atgtattaaa tcaatttatc actgtttaaa gctttgaata tctgcaatct ttgccaaggt acttttttat ttaaaaaaaa acataacttt gtaaatatta ccctgtaata ttatatatac ttaataaaac attttaagct attttgttgg gctatttcta ttgctgctac agcagaccac aagcacattt ctgaaaaatt taatttatta atgtattttt aagttgctta tattctaggt aacaatgtaa agaatgattt aaaatattaa ttatgaattt tttgagtata atacccaata agcttttaat tagagcagag ttttaattaa aagttttaaa tcagtc I. Preferred Compositions of the Present Invention

[0046] The preferred compositions of the present invention are morpholino oligonucleotides. As used herein, such oligonucleotides comprise a six member morpholino ring in lieu of the pentose sugar of DNA or RNA:

Deoxyribosyl Moiety Ribosyl Moiety Morpholino Moiety

[0047] Additionally, such morpholino oligonucleotides possess a non-ionic backbone, most preferably achieved by replacing the phosphorodiester bonds of DNA or RNA with phosphorodiamidate linkages:

[0048] Preferably, the sequences of the morpholino oligonucleotides of the present invention will be designed to be between 10-30 bases long, more preferably, 15-30 bases long, and still more preferably 20-25 bases long. Preferably, the sequence is designed to bind close to the CD47 ATG start codon, and to exhibit a Tm of 75°C - 115°C, more preferably, a Tm of 80°C -100°C, and still more preferably a Tm of 90°C -110°C. Preferably, the oligonucleotide will have a %GC content of 40-80%.

[0049] Although the invention particularly contemplates the treatment of humans and thus the ability to block the translation of human CD47, in preferred embodiments, the morpholino oligonucleotides of the present invention will additionally be capable of hybridizing to the CD47 R A or DNA of non-human species (especially murine, rodent, primate, canine, porcine or feline).

[0050] Consistent with the above indicated parameters, the present invention provides the following preferred morpholino oligonucleotides. Additionally, the invention contemplates further improved morpholino oligonucleotides that have been shortened to improve specificity and to increase their targeting to the start codon (to avoid potential for oligonucleotide failure due to downstream internal ribosome entry). The splice modifying oligonucleotides described below have better CG% and Tm characteristics and their activities can be assessed at the RNA level by RT-PCR.

[0051] Indeed, the present invention discloses morpholino oligonucleotides that are shorter than 25 bases and which exhibit decreased overall affinity relative to prior anti-CD47 morpholino oligonucleotides, while exhibiting increased target specificity). The translation-blocking morpholino oligonucleotides are targeted across the start codon. The splice-modifying morpholino oligonucleotides are targeting regions of the pre-mRNA with lower CG content than the 5'-UTR + start of coding region, leading to morpholino oligonucleotides that are likely to be more specific for their intended targets. The splice-modifying morpholino oligonucleotides are targeted to interfere with snRNP binding at intronic sites near the splice junctions.

[0052] These modifications are significant since mouse and human CD47 present difficult 5' untranslated region (5'-UTR) targets, as the CG contents of the target sequences are very high. The danger of a high-CG target is that subsequences of the oligonucleotide may be complementary to non-target RNA and that these subsequences might have sufficient affinity to bind and knock down the unintended non-targets. The new translation blocking oligonucleotide designs are shorted to decrease the probability of non-target interactions, though their GC contents are still above the ideal range. The translation-blocking oligonucleotides are targeted across the start codon, eliminating the chance of internal ribosome entry between the oligonucleotide target and the start codon.

[0053] The oligonucleotides of the present invention thus have less stable regions of self-complementarity than the prior existing technology, and are therefore expected to have higher antisense activity per molecule (because they are less prone to form homodimers or hairpins through the complementary sequences).

[0054] The splice-modifying oligonucleotides of the present invention have far lower GC contents and therefore are expected to be more specific, as they are less likely to have enough affinity for non-target RNA to bind with subsequences of the oligonucleotides. Unlike the translation blocking oligonucleotides, the molecular activity of the splice-modifying oligonucleotides can be monitored at the RNA level by RT-PCR by looking for either a shift in RT-PCR product mass on elimination of an exon from the mature transcript or by disappearance of the RT-PCR product if the removal of the exon triggers a frameshift and subsequent nonsense-mediated decay of the mature transcript.

A. Preferred Murine CD47 Favoring Morpholino

Oligonucleotide SEQ ID NO: 3

[0055] A preferred morpholino oligonucleotide of the present invention optimized for blocking the translation of the murine CD47 gene possesses the sequence: gccgccaagggccacatctcc (SEQ ID NO: 3), such that it will hybridize to that portion of the murine CD47 RNA or DNA having the sequence: ggagatgtggcccttggcggc (SEQ ID NO: 4), where the underlined atg sequence is the start codon of the murine CD47 gene. The predicted Tm of binding is 99.1 °C and the oligonucleotide has a GC content of 71%.

[0056] The oligonucleotide of SEQ ID NO: 3 will hybridize to that portion of human CD47 RNA or DNA having the sequence: ggaga tg tggc c cctggtagc (SEQ ID NO: 5), where the underlined atg sequence is the start codon of the murine CD47 gene and where the three mispairings are shown with wavy underline.

B. Preferred Human CD47 Favoring Morpholino

Oligonucleotide SEQ ID NO: 6

[0057] A preferred morpholino oligonucleotide of the present invention optimized for blocking the translation of the human CD47 gene possesses the sequence: ccacatctccgcgcccgccgc (SEQ ID NO: 6), such that it will hybridize to that portion of the murine CD47 RNA or DNA having the sequence: cccggcggcgcggagatgtgg (SEQ ID NO: 7), where the underlined atg sequence is the start codon of the murine CD47 gene, and the four mismatches are shown with wavy underline.

[0058] The oligonucleotide of SEQ ID NO: 6 will hybridize to that portion of human CD47 RNA or DNA having the sequence: gcggcgggcgcggagatgtgg (SEQ ID NO: 8), where the underlined atg sequence is the start codon of the murine CD47 gene. The predicted Tm of binding is 1 10.0 °C and the oligonucleotide has a GC content of 80%.

C. Preferred Murine CD47 Favoring Morpholino

Oligonucleotide SEQ ID NO: 9

[0059] A preferred morpholino oligonucleotide of the present invention optimized for modifying the splicing of the murine CD47 gene possesses the sequence: aacaggcaaactgtgtcacttaccc (SEQ ID NO: 9), such that it will hybridize to that portion of the murine CD47 RNA or DNA having a variant transcript that possesses the sequence: gggtaagtgacacagtttgcctgtt (SEQ ID NO: 10) (e.g. , a CD47 e2i2 morpholino oligonucleotide that hybridizes to the sequence ENSMUST00000084838). The predicted Tm of binding is 93.8 °C and the oligonucleotide has a GC content of 48%.

D. Preferred Human CD47 Favoring Morpholino

Oligonucleotide SEQ ID NO: 11

[0060] A preferred morpholino oligonucleotide of the present invention optimized for modifying the splicing of the human CD47 gene possesses the sequence: gctttcatagaagtcttaccaacac (SEQ ID NO: 11), such that it will hybridize to that portion of the human CD47 RNA or DNA having a variant transcript that possesses the sequence: gtgttggtaagacttctatgaaagc (SEQ ID NO: 12) (e.g. , a CD47 morpholino oligonucleotide that hybridizes to the sequence ENST00000355354). The predicted Tm of binding is 83.9 °C and the oligonucleotide has a GC content of 40%. II. Uses of the Compositions of the Present Invention

A. Radioprotection and Radiomitigation

[0061] The morpholino oligonucleotides of the present invention have particular utility in providing radioprotection to provide individuals with protection from a concurrent or from a future exposure to radiation or to a DNA damaging agent (especially an exposure occurring within 96 hours, within 72 hours, within 48 hours, within 24 hours or more imminently, of treatment).

[0062] Additionally, the morpholino oligonucleotides of the present invention have particular utility in providing radiomitigation to provide individuals with protection from the consequences of a prior exposure to radiation or to a DNA damaging agent (especially an exposure occurring within 96 hours, within 72 hours, within 48 hours, within 24 hours or more imminently, of treatment).

[0063] Preferably, such protection will decrease the consequence of such past, concurrent, or future exposure by 20%, by 50%, by 80%, by 90%, by 99.5%, by 99.9%) or by 2, 3, 4, 5, 6 or more orders of magnitude relative to the consequence experienced by an untreated individual.

[0064] The most important acute side effect of radiation poisoning is damage to the bone marrow. The bone marrow produces all the normal blood cells, and therefore a high dose of radiation can lead to low blood counts of red cells, platelets and white blood cells (resulting in anemia, bleeding, and compromised immunity). In one embodiment, radiomitigation can be achieved by administering the morpholino oligonucleotides of the present invention to bone marrow (in vivo), or to cells removed from bone marrow (ex vivo). However, other means of administration (especially oral, intravenous or subcutaneous routes of administration) are capable of delivering such agents to bone marrow and of providing radiomitigation (see, e.g., Johnson, S.M. et al. (2010) "Mitigation Of Hematologic Radiation Toxicity In Mice Through Pharmacological Quiescence Induced By CDK4/6 Inhibition," J. Clin. Invest. 120(7):2528-2536; Amantana, A. et al. (2005) "Pharmacokinetics And Biodistribution Of Phosphorodiamidate Morpholino Antisense Oligomers^'1'' Current Opinion in Pharmacology 5:550-555; Maxhimer, J.B. et al. (2009) "Radioprotection in Normal Tissue and Delayed Tumor Growth by Blockade of CD47 Signaling " Sci. Transl. Med. 1(3): 3ra7. doi: 10.1126/scitranslmed.3000139; pages 1-13).

[0065] Significantly, since the inhibition of CD47 activation that is achieved by the compositions of the present invention relate to a fundamental mechanism of DNA damage, the compositions and methods of the present invention are effective in providing radioprotection regardless of the nature of the radiation involved (i.e., isotope and/or wavelength independent).

B. Treatment of Cancer

[0066] Radiation therapy is currently administered to approximately 50% of newly diagnosed cancer patients (Isenberg, J.S. et al. (2008) "Thrombospondin-I and CD47 Limit Cell and Tissue Survival of Radiation Injury " Amer. J. Pathol. 173(4) 1100- 1112). Such administration reflects the recognition that radiation-induced DNA damage compromises the ability of cells to undergo mitosis, and thus that the tissues most sensitive to effects of radiation will be those, such as cancer cells, that are undergoing rapid proliferation. Several mechanisms contribute to radiation-induced cell death including mitotic death, apoptosis, and cell cycle arrest. The mechanism and extent of cell death depend on cell type, cell environment, and the radiation dose.

[0067] Unfortunately, the use of radiation therapy has been found to affect not only the targeted cancer cells but also healthy cells that did not receive direct radiation ("bystander effects") (Blyth, B.J. et al. (June 2011) "Radiation-Induced Bystander Effects: What Are They, and How Relevant Are They to Human Radiation Exposures?^ Radiat. Res. (doi: 10.1667/RR2548.1); Fischer, P. et al. (May 2011) The Bystander-Effect: A Meta-Analytic Review On Bystander Intervention In Dangerous And Non-Dangerous Emergencies " Psychol. Bull. (doi: 10.1037/a0023304); Baskar, R. (2010) "Emerging Role Of Radiation Induced Bystander Effects: Cell Communications And Carcinogenesis " Genome Integr. 1(1): 13 (1-8); Burdak-Rothkamm, S. et al. (2009) "New Molecular Targets In Radiotherapy: DNA Damage Signalling And Repair In Targeted And Non-Targeted Cells," Eur. J. Pharmacol. 625(1-3): 151-155; Rzeszowska-Wolny, J. et al. (2009) "Ionizing Radiation-Induced Bystander Effects, Potential Targets For Modulation Of Radiotherapy " Eur. J. Pharmacol. 625(1-3): 156-64; Morgan, W.F. et al. (2009) "Non-Targeted Effects Of Ionizing Radiation: Implications For Risk Assessment And The Radiation Dose Response Profile," Health Phys. 97(5):426-432; Munro, A.J. (2009) "Bystander Effects And Their Implications For Clinical Radiotherapy," J. Radiol. Prot. 29(2A):A133-A142). As a consequence, direct radiation damage of cancer tissue can lead to the death of otherwise healthy non-cancerous tissue (Prise, K.M. et al. (2005) "New Insights On Cell Death From Radiation Exposure," Lancet Oncol 6:520-528). It has therefore been necessary in the past to limit the dosage of radiation provided to a tumor in order to avoid causing such damage to healthy tissue (Isenberg, J.S. et al. (2008) "Thrombospondin-I and CD47 Limit Cell and Tissue Survival of Radiation Injury," Amer. J. Pathol. 173(4) 1100-1112).

[0068] The present invention addresses this problem by providing a means for suppressing CD47-mediated cell death, and thus provides a therapy for cancers including: breast cancer, prostate cancer, gastric cancer, lung cancer, stomach cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, ovarian cancer, oral cavity cancer, pharyngeal cancer, esophageal cancer, laryngeal cancer, bone cancer, skin cancer, melanoma, uterine cancer, testicular cancer, bladder cancer, kidney cancer, brain cancer, glioblastoma, thyroid cancer, lymphoma, myeloma, and leukemia.

C. Treatment of Cardiovascular Disease

[0069] The morpholino oligonucleotides of the present invention also have utility in the treatment of cardiovascular disease. Also contemplated are uses of provided therapeutic compounds and compositions in ameliorating reperfusion injury following cardiac ischemia (Ml/heart attack), or extracorporeal oxygenation (bypass) during heart surgery, heart transplant lung or heart-lung transplants.

[0070] Without intending to be limited to any mechanism of action, it is believed that such utility reflects the ability of such oligonucleotides to inhibit the CD-47 mediated inhibition of nitric oxide regulation of the vasculature.

[0071] As discussed above, under hypoxic conditions such as ischemia or myocardial infarction, nitric oxide (NO) is produced and acts to promote the relaxation of vascular smooth muscle cells and to thereby increase blood vessel diameter and blood flow (Isenberg, J.S. et al. (2008) "Thrombospondin-1 is a Central Regulator of Nitric Oxide Signaling in Vascular Physiology " Cell. Mol. Life Sci. 65(5):728-742; Gladwin, M.T. et al. (2006) 'Nitrite As A Vascular Endocrine Nitric Oxide Reservoir That Contributes To Hypoxic Signaling, Cytoprotection, And Vasodilation " Am. J. Physiol. Heart Circ. Physiol. 291 :H2026-H2035; Shiva, S. et al. (2007) "Deoxymyoglobin Is A Nitrite Reductase That Generates Nitric Oxide And Regulates Mitochondrial Respiration,"Circ. Res. 100:654-661; Rassaf, T. et al. (2007) "Nitrite Reductase Function Of Deoxymyoglobin: Oxygen Sensor And Regulator Of Cardiac Energetics And Function," J. Circ. Res. 100: 1749-1754)). The produced nitric oxide

D. Treatment of inflammation

[0072] Elevated tissue expression of thrombospondin-1 has been found to be a characteristic of several chronic inflammatory diseases, including diabetes and atherosclerosis (Isenberg, J.S. et al. (2008) "Thrombospondin-1 is a Central Regulator of Nitric Oxide Signaling in Vascular Physiology," Cell. Mol. Life Sci. 65(5):728-742; Favier, J. et al. (2005) "Critical Over expression Of Thrombospondin I In Chronic Leg Ischaemia," J. Pathol. 207:358-366; Riessen, R. et al. (1998) "Immunolocalization Of Thrombospondin-1 In Human Atherosclerotic And Restenotic Arteries " Am. Heart J. 135:357-364; Roth, J.J. et al. (1998) "Thrombospondin- 1 Is Elevated With Both Intimal Hyperplasia And Hypercholesterolemia," Surg. Res. 74: 11-16; Hida, K. et al. (2000) "Identification Of Genes Specifically Expressed In The Accumulated Visceral Adipose Tissue Of OLETF Rats," J. Lipid Res. 41 : 1615- 1622).

[0073] Since the oligonucleotides of the present invention disrupt CD47 activity, they have therapeutic utility in the prevention and management of inflammation.

E. Treatment of CNS Disorders

[0074] Since the oligonucleotides of the present invention disrupt CD47 activity, they also have therapeutic utility in the prevention and management of CNS disorders, such as multiple sclerosis, meningitis, encephalitis, stroke, other cerebral traumas, inflammatory bowel disease including ulcerative colitis and Crohn's disease, myasthenia gravis, lupus, rheumatoid arthritis, asthma, acute juvenile onset diabetes, AIDS dementia, atherosclerosis, nephritis, retinitis, atopic dermatitis, psoriasis, myocardial ischemia and acute leukocyte-mediated lung injury.

II. Administration of the Compositions of the Present Invention

[0075] Methods are thus disclosed herein for promoting blood flow in an area in a subject (e.g., a patient or recipient of the therapy) who has or is at risk for developing ischemia, for instance during or following surgery, burn injury, a graft, peripheral vascular disease, amputation, coronary artery disease, stroke, thrombosis, a clot, chronic vascular obstruction or vasculopathy (e.g., secondary to diabetes, hypertension, or peripheral vascular disease), cerebral ischemia, a wound, and so forth. The provided methods are useful in the treatment of various diseases and conditions, including but not limited to treatment of donor organs before and after transplantation; reattachment of severed extremities, body parts or soft tissues; pulmonary hypertension (adult or neonate); sickle cell disease; neointimal hyperplasia or restenosis (following angioplasty or stenting); primary burn care (before skin grafting); kidney disease (for instance, to increase kidney circulation); pre-eclampsia; erectile dysfunction; asthma or adult respiratory distress syndrome; Alzheimer's and other dementias secondary to compromised cranial blood flow. Methods are also disclosed herein for promoting blood flow and increasing tissue perfusion in a tissue, such as in an elderly subject with a degenerative disorder, such as in the brain of an elderly subject with dementia or Alzheimer's disease. The methods include introducing a therapeutically effective amount of a morpholino oligonucleotide whose sequence is selected so as to be capable of selectively and hybridizing to the CD47 gene or to its transcribed RNA, and to thereby block translational synthesis of CD47 protein or alter the splicing of the CD47 RNA so as to produce non-functional or impaired functional variant CD47 proteins.

[0076] The therapeutic oligonucleotides of the p-resent invention may be advantageously administered in conjunction with a nitric oxide (NO) donor or precursor, such as isosorbide dinitrate, Bidil or L-arginine, or NO generating topical agents. Such therapeutic agents also may be administered in conjunction with agents that act upon soluble guanylyl cyclase to activate the enzyme and with agents that act to inhibit cyclic nucleotide phosphodiesterases (Viagra.RTM., Levitra.RTM., Cialis.RTM., for instance).

[0077] In some embodiments, the above-described inhibitory morpholino oligonucleotide is administered locally to an affected area, for example by direct topical administration to a wound or other lesion in which radioprotection, neovascularization, etc. is desired, or is incorporated into a vascular stent or other implant device and placed directly in a diseased or healthy blood vessel, or is parenterally directed to an affected area, such as an ischemic extremity or an area exposed to radiation. For subjects with peripheral vascular disease, administration is, for example, preferably by direct topical administration to a wound, or by intraarterial, intravenous, subcutaneous, or intramuscular injection into the affected limb. Efficacy of the treatment is shown, for example, by a regression of symptoms, for example, a lessening of cramping in the leg or arm, or a lessening of claudication, numbness, tingling, weakness, or pain, or healing of skin ulcers on the limb. An improvement in vascular function is also demonstrated, for example, by increased skin temperature or a color change in the skin of the limbs.

[0078] A therapeutic agent can also be administered by intra- ventricular injection for degenerative disease of the brain, such as to an elderly subject with Alzheimer's disease or dementia.

[0079] Additionally, in some embodiments the therapeutic agents may be incorporated in implantable devices, such as vascular stents placed directly in diseased blood vessels in the coronary, cerebral or peripheral circulation, for instance to provide slow release of the compound, thereby providing regional sustained release of the therapeutic agents.

[0080] For subjects with peripheral artery disease (and other systemic and arterial diseases), administration is, for example, preferably by intra-arterial (particularly intracoronary), or intrapericardial injection. In some embodiments, the therapeutic agent is administered systemically, such as by intravenous injection. Additionally, in some embodiments the therapeutic agents may be incorporated into or on an implantable device, such as vascular stents placed directly in diseased blood vessels in the coronary or cerebral circulation, and undergo slow release providing regional sustained release of the therapeutic agents. Efficacy of treatment is demonstrated, for example, by a regression of symptoms, for example chest pressure or pain.

[0081] For subjects with a wound such as a burn or a graft, administration is, for example, preferably by subcutaneous or intravenous injection, by direct injection of the wound or burn or graft bed, or by topical application. Efficacy of the treatment is determined, for example, by an improvement in wound healing.

[0082] Administration may begin whenever a subject has developed, or is at risk for developing a condition (e.g., ischemia, when radiation damage has occurred or is anticipated, when a wound, burn, graft, transplant or the like has occurred, or when symptoms of reduced blood flow to the brain, heart, or one or more limbs are present, such as chest or limb pain, or neurological symptoms, such as dizziness, confusion, loss of speech, or loss of mobility).

[0083] The therapeutic molecules of the present invention can be administered in a single dose, or in multiple doses, for example daily, weekly, every two weeks, or monthly during a course of treatment. Additionally, the therapeutic agents may be incorporated into or on implantable constructs or devices, such as vascular stents, for sustained regional or local release.

[0084] Alternatively, the morpholino oligonucleotides of the present invention can be administered directly, for instance in the form of topical applied liquids or creams or via direct injection into tissues. Additionally, such agents can be incorporated into the irrigation fluid such as that routinely used to wash wound beds prior to closure. In this form, the therapeutic agents would be placed specifically were needed to maximize blood flow, angiogenesis and wound healing. The direct application of such morpholino oligonucleotides a would also provide therapeutic benefit for burn patients undergoing skin grafts, by insuring improved blood flow to the healing graft.

[0085] The therapeutic agents described herein can also play a role in decreasing the overall size of the burn wound. Direct injection into the margins of the burn wound would encourage blood flow to the ischemic wound areas and maximize tissue survival.

[0086] In the case of skin grafts and burn wounds, controlled release of any of the described compounds over extended periods of time may be of significant benefit in healing.

[0087] Methods are provided herein for increasing tissue perfusion in elderly subjects. Thus, the disclosed methods are of use in any subject, such as a mammalian subject, who has passed middle age. In one embodiment, an elderly mammalian subject is a subject that has survived more than two-thirds of the normal lifespan for that mammalian species. In a further embodiment, for humans, an aged or elderly subject is more than about 65 years of age, such as a subject of more than about 70 years of age, more than about 75 years of age, or more than about 80 years of age. In yet another embodiment, for mice, the disclosed methods are of use in animals that are from about 14 to about 18 months of age. One of skill in the art can readily distinguish the elderly subject for a specific mammalian species, based on the average lifespan for that species. It will be recognized that methods disclosed herein will also be effective in increasing blood flow secondary to tissue trauma or vascular disease regardless of the age of the subject

[0088] In several embodiments, providing a nitric oxide source at the same time one eliminates or reduces effects of TSPl can enhance tissue survival to ischemia. The therapeutic application of combination therapy employing an exogenous source of nitric oxide (such as isosorbide dinitrate) and TSPl or CD47 blockade or suppression (e.g., with antibody or morpholino or small molecule inhibitors) can be used to enhance blood flow and angiogenesis to ischemic tissue and increase blood flow, tissue survival and blood flow.

[0089] Furthermore, the morpholino oligonucleotide therapeutic agents of the present invention may also be used in combination with other therapeutic agents, for example, pain relievers, anti-inflammatory agents, antihistamines, and the like, whether for the conditions described or some other condition. By way of example, the additional agent is one or more selected from the list consisting of an antibiotic (e.g., penicillin), hydroxyurea, butyrate, clotrimazole, arginine, or a phosphodiesterase inhibitor (such as sildenafil). The therapeutic agents of the present invention can be used in combination with any pharmaceutical composition of use to treat a disorder.

[0090] The therapeutic compounds described herein may be formulated in a variety of ways depending on the location and type of disease to be treated or prevented in the subject. Pharmaceutical compositions are thus provided for both local use at or near an affected area and for systemic use (in which the agent is administered in a manner that is widely disseminated via the cardiovascular system). This disclosure includes within its scope pharmaceutical compositions including at least one morpholino oligonucleotide formulated for use in human or veterinary medicine. While such molecules typically will be used to treat human subjects, they may also be used to treat similar or identical diseases in other vertebrates, such other primates, dogs, cats, horses, and cows.

[0091] Pharmaceutical compositions that include at least one such morpholino oligonucleotide as described herein as an active ingredient may be formulated with an appropriate solid or liquid carrier, depending upon the particular mode of administration chosen. Additional active ingredients include, for example, nitric oxide donors, nitrovasodilators, activators of the enzyme soluble guanylylcyclase, or cGMP phosphodiesterase inhibitors.

[0092] A suitable administration format may best be determined by a medical practitioner for each subject individually. Various pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, for example, Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang and Hanson, J. Parenteral Sci. TechnoL, Technical Report No. 10, Supp. 42: 2S, 1988. VIVO-MORPHOLINOS™ (GeneTools, LLC) may be employed to facilitate or accomplish delivery of the oligonucleotides (see, e.g., Morcos, P.A. et al. (2008) "Vivo -Morpholino s: A Non-Peptide Transporter Delivers Morpholinos Into A Wide Array Of Mouse Tissues " BioTechniques 45(6):616-626).

[0093] The dosage form of the pharmaceutical composition will be determined by the mode of administration chosen. For instance, in addition to injectable fluids, inhalational, topical, opthalmic, peritoneal, and oral formulations can be employed. Inhalational preparations can include aerosols, particulates, and the like. In general, the goal for particle size for inhalation is about 1 μιη or less in order that the pharmaceutical reach the alveolar region of the lung for absorption. Oral formulations may be liquid (for example, syrups, solutions, or suspensions), or solid (for example, powders, pills, tablets, or capsules). For solid compositions, conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. Actual methods of preparing such dosage forms are known, or will be apparent, to those of ordinary skill in the art.

[0094] The compositions or pharmaceutical compositions of the present invention can be administered by any route, including parenteral administration, for example, intravenous, intramuscular, intraperitoneal, intrasternal, or intra-articular injection or infusion, or by sublingual, oral, topical, intra-nasal, ophthalmic, or transmucosal administration, or by pulmonary inhalation. When the active compounds are provided as parenteral compositions, for example, for injection or infusion, they are generally suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. Useful buffers include sodium citrate-citric acid and sodium phosphate- phosphoric acid, and sodium acetate/acetic acid buffers. A form of repository or depot slow release preparation may be used so that therapeutically effective amounts of the preparation are delivered into the bloodstream over many hours or days following transdermal injection or delivery.

[0095] The therapeutic compounds of the present invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release formulations include suitable polymeric materials (such as, for example, semipermeable polymer matrices in the form of shaped articles, for example, films, or mirocapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt). Sustained-release compounds may be administered by intravascular, intravenous, intra-arterial, intramuscular, subcutaneous, intra- pericardial, or intra-coronary injection. Administration can also be oral, rectal, parenteral, intracisternal, intravaginal, intraperitoneal, topical (as by powders, ointments, gels, drops or transdermal patch), buccal, or as an oral or nasal spray.

[0096] Preparations for administration can be suitably formulated to give controlled release of the therapeutic agent(s). For example, the pharmaceutical compositions may be in the form of particles comprising a biodegradable polymer and/or a polysaccharide jellifying and/or bioadhesive polymer, an amphiphilic polymer, an agent modifying the interface properties of the particles and a pharmacologically active substance. These compositions exhibit certain biocompatibility features that allow a controlled release of the active substance. See, for example, U.S. Pat. No. 5,700,486.

[0097] In some embodiments, therapeutic agent(s) of the present invention are delivered by way of a pump (see Sefton, CRC Crit. Ref Biomed. Eng. 14:201, 1987; Buchwald et al, Surgery 88:507, 1980; Saudek et al, N. Engl. J. Med. 321 :574, 1989) or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The key factor in selecting an appropriate dose is the result obtained, as measured by increases or decreases in angiogenesis, or by other criteria for measuring control or prevention of disease, as are deemed appropriate by the practitioner. Other controlled release systems are discussed in the review by Langer (Science 249: 1527-1533, 1990).

[0098] In another aspect of the disclosure, therapeutic agent(s) are delivered by way of an implanted pump, described, for example, in U.S. Pat. No. 6,436,091; U.S. Pat. No. 5,939,380; and U.S. Pat. No. 5,993,414. Implantable drug infusion devices are used to provide subjects with a constant and long term dosage or infusion of a drug or any other therapeutic agent. Essentially, such device may be categorized as either active or passive.

[0099] Active drug or programmable infusion devices feature a pump or a metering system to deliver the drug into the patient's system. An example of such an active drug infusion device currently available is the Medtronic SynchroMed.TM. programmable pump. Such pumps typically include a drug reservoir, a peristaltic pump to pump the drug out from the reservoir, and a catheter port to transport the pumped out drug from the reservoir via the pump to a patient's anatomy. Such devices also typically include a battery to power the pump, as well as an electronic module to control the flow rate of the pump. The Medtronic SynchroMed.TM. pump further includes an antenna to permit the remote programming of the pump.

[00100] Passive drug infusion devices, in contrast, do not feature a pump, but rather rely upon a pressurized drug reservoir to deliver the drug. Thus, such devices tend to be both smaller as well as cheaper as compared to active devices. An example of such a device includes the Medtronic IsoMed.TM.. This device delivers the drug into the patient through the force provided by a pressurized reservoir applied across a flow control unit.

[00101] The implanted pump can be completely implanted under the skin of a subject, thereby negating the need for a percutaneous catheter. These implanted pumps can provide the patient with therapeutic agent(s) at a constant or a programmed delivery rate. Constant rate or programmable rate pumps are based on either phase-change or peristaltic technology. When a constant, unchanging delivery rate is required, a constant-rate pump is well suited for long-term implanted drug delivery. If changes to the infusion rate are expected, a programmable pump may be used in place of the constant rate pump system. Osmotic pumps may be much smaller than other constant rate or programmable pumps, because their infusion rate can be very low. An example of such a pump is described listed in U.S. Pat. No. 5,728,396.

[00102] The therapeutic agents may also be delivered passively and in sustained fashion as part of and incorporated into implantable devices, such as vascular stents which can be placed directly into diseased blood vessels through several standard approaches, including direct surgical insertion or percutaneoulsy with angiographic control.

[00103] For oral administration, the pharmaceutical compositions of the present invention can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (for example, pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (for example, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (for example, magnesium stearate, talc or silica); disintegrants (for example, potato starch or sodium starch glycolate); or wetting agents (for example, sodium lauryl sulphate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (for example, lecithin or acacia); non-aqueous vehicles (for example, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (for example, methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate.

[00104] For administration by inhalation, the therapeutic compounds of the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[00105] For topical administration, the therapeutic compounds of the present invention can be, for example, mixed with a liquid delivery agent for administration locally. The agents used therapeutically (such as peptides, antibodies and morpholinos) are readily soluble or suspendable in water and saline, and as such these would be useful for delivery since water or saline do not cause adverse biological tissue effects. This allows sufficiently high doses to be administered locally or systemically, without secondary toxicity from the delivery vehicle.

[00106] By way of example, in the treatment of burns, agents can be given by direct injection into the wound bed or topically dissolved in saline as a spray to the burn area, to skin grafts and/or to graft wound beds. They may also be mixed directly into antibiotic creams used to treat burns, such as bacitracin or silver sulfadine, or incorporated in a manner allowing release into dressing and bandaging materials applied to wounds, grafts or burns.

[00107] Pharmaceutical compositions that comprise at least one therapeutic agent as described herein as an active ingredient will normally be formulated with an appropriate solid or liquid carrier, depending upon the particular mode of administration chosen. The pharmaceutically acceptable carriers and excipients useful in this disclosure are conventional. For instance, parenteral formulations usually comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like. Excipients that can be included are, for instance, proteins, such as human serum albumin or plasma preparations. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.

[00108] All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

Claims

What Is Claimed Is:

Claim 1. An agent that inhibits interaction between thrombospondin-1 (TSP1) and CD47 for use in a method of protecting animal tissue from damage caused by radiation exposure, comprising contacting the tissue with a therapeutically effective amount of the agent.

Claim 2. The agent for use in the method of claim 1, which agent is employed in a method of protecting a subject exposed to a radioactive substance or ionizing radiation, comprising contacting tissue of the subject with the therapeutically effective amount of the agent.

Claim 3. The agent for use in the method of claim 1 or 2, wherein said contacting is performed at least one of before, during or after exposure to radiation, and preferably, wherein the agent is administered within two weeks prior to exposure to radiation, during radiation exposure, and/or within two weeks following radiation exposure, or wherein the agent is administered within four days prior to radiation exposure, during radiation exposure, and/or within about one day following radiation exposure.

Claim 4. The agent for use in the method of any one of claims 1-3, wherein the radiation comprises an acute or chronic dose of ionizing or nonionizing radiation and preferably, wherein the ionizing radiation (i) results from nuclear fission or fusion, or (ii) comprises X-rays, or (iii) comprises radionuclides.

Claim 5. The agent for use in the method of claim 1-4, which agent is employed in a method of enhancing the therapeutic window for radiotherapy in a subject, comprising contacting tissue of the subject with the therapeutically effective amount of the agent prior to the radiotherapy.

Claim 6. The agent for use in the method of claim 1-5, wherein the radiation exposure comprises diagnostic X-rays, radiation therapy, a CAT-scan, a mammogram, a radionuclide scan, or an interventional radiological procedure under CT or fluoroscopy guidance.

Claim 7. The agent for use in the method of any one of claims 1 to 6, wherein the radiation exposure comprises tissue -incorporated radionuclides from ingestion of contaminated food or water, non-medical or unintentional exposure to ionizing radiation.

Claim 8. An agent that inhibits interaction between TSP1 and CD47 for use in a method of increasing tumor ablation in a subject undergoing radiotherapy, comprising contacting tissue of the subject with a therapeutically effective amount of the agent, which agent comprises: an oligonucleotide comprising at least about 15 contiguous bases and that hybridizes to the mRNA of CD47 with 0, 1, 2, 3, 4, or 5 mismatches.

Claim 9. The agent for use in the method of any one of claims 1-8, wherein the agent is administered topically, buccally, intraocularly, orally, subcutaneously, intramuscularly, intravenously, intraarterially, transdermally, intranasally, rectally, peritoneally, or by inhalation.

Claim 10. The agent for use in the method of any one of claims 1-9, wherein inhibiting interaction between TSP1 and CD47 comprises one or more of the following:

(a) inhibiting expression of CD47;

(b) removing endogenous CD47;

(c) blockading interaction between endogenous TSP1 and CD47.

Claim 11. The agent for use in the method of any one of claims 1-10, wherein (i) the agent comprises an oligonucleotide morpholino comprising at least about 15 contiguous bases and that hybridizes to the mRNA of CD47 under high stringency conditions, and preferably, wherein the oligonucleotide is a morpholino and preferably, wherein the morpholino comprises the sequence shown in SEQ ID NO: 6, 11, 3 or 9.

Claim 12. An agent for use in the above claims to render cancer sensitive to radiation killing by any form of therapeutically applied form of radiation.

Claim 13. An agent for use in the above claims that specifically is employed in the course of radiation therapy that render soft tissue carcinoma more sensitive to radiation such that lower total radiation is required to achieve cancer killing.

Claim 14. An agent for use in the above claims that specifically is employed to render blood borne cancers more susceptible to radiation killing.

Claim 15. An agent for use in the above claims that inhibits the interaction of thrombospondin-1 (TSP-1) and CD47 for use in a method of increasing tissue perfusion in a subject, comprising selecting a subject in need of increased tissue perfusion; and administering to the subject a therapeutically effective amount of the agent, thereby increasing tissue perfusion in the subject.

Claim 16. One or more agents that inhibit function or expression of CD47 for use in method comprising selective application to a subject of the one or more agents, which method: improves tissue or organ survival in the subject; treats or ameliorates peripheral vascular disease or myocardial ischemia in the subject; improves blood flow in the subject;

(a) improves transplant organ or tissue survival in the subject;

(b) improves wound healing of acute and/or chronic wounds;

(c) improves healing of surgical wounds;

(d) improves healing of traumatic injuries;

(e) improves burn survival or recover in the subject;

(f) improves skin graft survive in the subject; and/or

(g) improves survival of reattached extremities and body parts.

Claim 17. An agent that influences interaction between TSP-1 and CD47 for use in a method of controlling blood pressure in a subject.

Claim 18. The agent or agents for use of in the above claims, wherein the subject has diabetes.

Claim 19. An agent that inhibits the interaction of thrombospondin-1 (TSP-1) and

CD47 for use in a method of treating tissue necrosis resulting from ischemia in an elderly subject with atherosclerotic vascular disease or age-related vasculopathy, comprising: selecting an elderly subject with necrosis in a tissue, wherein the subject has atherosclerotic disease or age-related vasculopathy; and administering to the subject a therapeutically effective amount of the agent, thereby treating the tissue necrosis.

Claim 20. The agent for use of in the above claims, wherein the elderly subject:

has atherosclerosis, has a graft; has had a myocardial infarction; has a vasculopathy, has Alzheimer's; has dementia; or any combination of two or more thereof.