WO2005054426A2 - Angiogenesis affecting polypeptides, proteins, and compositions, and methods of use thereof - Google Patents
Angiogenesis affecting polypeptides, proteins, and compositions, and methods of use thereof Download PDFInfo
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- WO2005054426A2 WO2005054426A2 PCT/SE2004/001814 SE2004001814W WO2005054426A2 WO 2005054426 A2 WO2005054426 A2 WO 2005054426A2 SE 2004001814 W SE2004001814 W SE 2004001814W WO 2005054426 A2 WO2005054426 A2 WO 2005054426A2
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- angiogenesis
- embryos
- nucleic acid
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Definitions
- Angiogenesis affecting polypeptides, proteins, and compositions, and methods of use thereof.
- the invention relates to polypeptides and proteins encoded thereby which are involved in vasculogenesis and/or angiogenesis. These agents may be targeted when producing materials and methods used in the diagnosis and therapy of angiogenesis-related conditions.
- the invention flirther relates to such diagnostic and therapeutic methods and agents.
- vasculogenesis the development of an interactive vascular system comprising arteries and veins
- angiogenesis the generation of new blood vessels
- angiogenesis is limited in a normal adult to the placenta, ovary, endometrium, and sites of wound healing.
- Angiogenesis or its absence, plays an important role in the maintenance of a variety of pathological states. Some of these states are characterized by neovascularization, e.g., cancer, diabetic retinopathy, glaucoma, and age related macular degeneration. Others, e.g., stroke, infertility, heart disease, ulcers, and scleroderma, are diseases of angiogenic insufficiency.
- Angiogenesis has a number of stages (see, e.g., Zhu and Witte, Invest New Drugs 17:195-212, 1999).
- the early stages of angiogenesis include endothelial cell protease production, migration of cells, and proliferation. The early stages also appear to require some growth factors, with NEGF, TGF-A and selected chemokines all putatively playing a role.
- Later stages of angiogenesis include population of the vessels with mural cells (pericytes or smooth muscle cells), basement membrane production, and the induction of vessel bed specializations.
- the final stages of vessel formation include what is known as remodelling wherein a forming vasculature becomes a stable, mature vessel bed. Thus, the process is highly dynamic, often requiring coordinated spatial and temporal waves of gene expression.
- the complex angiogenesis process is subject to disruption through interference with one or more critical steps, and numerous disease states can result from or be exacerbated by the disruption.
- Unregulated angiogenesis can cause or worsen disease, for example, ocular neovascularization has been implicated as the most common cause of blindness and underlies the pathology of approximately 20 eye diseases.
- ocular neovascularization has been implicated as the most common cause of blindness and underlies the pathology of approximately 20 eye diseases.
- ocular neovascularization has been implicated as the most common cause of blindness and underlies the pathology of approximately 20 eye diseases.
- ocular neovascularization has been implicated as the most common cause of blindness and underlies the pathology of approximately 20 eye diseases.
- ocular neovascularization has been implicated as the most common cause of blindness and underlies the pathology of approximately 20 eye diseases.
- ocular neovascularization has been implicated as the most common cause of blindness and underlies the pathology
- the present invention discloses ten nucleic acid sequences and associated proteins which have key roles in vasculogenesis and/or angiogenesis.
- One object of the present invention is to present approaches for using the ten novel factors as molecular targets for therapeutic intervention in angiogenesis-related disease states. It is a further object of the present invention to provide materials and methods that can be used to screen compounds for the ability to modulate angiogenesis or angiogenesis-related conditions.
- Therapeutics specifically targeting the sequences and proteins identified herein are also provided as agents or compositions which modulate vasculogenesis or angiogenesis.
- an isolated nucleic acid molecule according to any one of SEQ ID NO:s 2, 4, 7, 9, 12, 14, 17, 19, 21, 24, 26, 29, 31, 34, 36, 39, 41, 44, 46, 49, and 51 or a fragment or analogue thereof is provided which has the ability to stimulate or inhibit at least one biological activity selected from the group consisting of vasculogenesis, angiogenesis, vascular permeability, endothelial cell proliferation, endothelial cell differentiation, endothelial cell migration, and endothelial cell survival, or an isolated nucleic acid molecule which hybridizes to one of the foregoing sequences under stringent conditions
- the invention is also directed to isolated nucleic acid molecules which hybridizes to a compliment of a nucleic acid molecule described above, and an isolated siRNA molecule of at least 19 base pairs targeted to an isolated nucleic acid molecule described above.
- an expression vector comprising one of the novel nucleic acids.
- the nucleic acid may be operatively associated with a regulatory nucleic acid controlling the expression of the polypeptide encoded by the nucleic acid.
- the invention further comprises host cells genetically engineered to contain a nucleic acid as described above, or transfected by an expression vector described above.
- a method of treating an angiogenesis- related condition in a cell, group of cells, or organism comprising administering an expression vector as described above to the cell, group of cells, or organism.
- the invention further comprises antibodies with specific reactivity to the nucleic acid molecules described above.
- the antibodies may be polyclonal or monoclonal and may further comprise detectable labels, such as fluorescent labels.
- a transgenic, non-human animal which has been genetically engineered to contain a transgene comprising a nucleic acid as described above, and animals which contain and express the transgene.
- a pharmaceutical composition which comprises a nucleic acid sequence as described above.
- the compound may be administered to a cell, group of cells, or organism to affect vasculogenesis or angiogenesis.
- the effect may be to increase or decrease vasculogenesis or angiogenesis, and the method may be employed where the cells, group of cells, or organism has an angiogenesis-related disorder.
- Such angiogenesis-related disorders include cancer, retinopathy, macular degeneration, corneal ulceration, stroke, ischemic heart disease, infertility, ulcers, scleradoma, wound healing, ischemia, ischemic heart disease, myocardial infarction, myocardosis, angina pectoris, unstable angina, coronary arteriosclerosis, arteriosclerosis obliterans, Berger's disease, arterial embolism, arterial thrombosis, cerebrovascular occlusion, cerebral infarction, cerebral thrombosis, cerebral embolism, rubeosis prohferative vitreoretinopathy, chronic inflammation, inflammatory bowel disease, psoriasis, sarcoidosis, and rheumatoid arthritis.
- an isolated polypeptide comprising a sequence of amino acids substantially corresponding to the amino acid sequence in any one of SEQ ID NO:s 3, 5, 8, 10, 13, 15, 18, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 47, 50, and 52 or a fragment or analogue thereof is provided which has the ability to affect angiogenesis in a cell, a group of cells, or an organism.
- the invention further comprises host cells genetically engineered to express a polypeptide as described above, as well as antibodies specifically reactive with the polypeptides.
- the antibody may be polyclonal or monoclonal, and may further comprise a detectable label such as fluorescence.
- a transgenic, non-human animal which has been genetically engineered to contain a transgene comprising a nucleic acid which encodes a polypeptide as described above, and animals that contain and express the transgene.
- the invention further provides pharmaceutical compositions comprising an isolated polypeptide as described above.
- the pharmaceutical composition may be administered to a cell, group of cells, or organism in order to affect vasculogenesis or angiogenesis therein.
- Vasculogenesis or angiogenesis may be increased or decreased.
- the cell, group of cells, or organism may have an angiogenesis-related disorder. Representative angiogenesis-related disorders are noted above.
- a method of detecting an angiogenesis- related transcript in a cell in a patient comprising contacting a biological sample from the patient with a polynucleotide that selectively hybridizes to a sequence at least 80% identical to a sequence according to any one of SEQ ID NO:s 2, 4, 7, 9, 12, 14, 17, 19, 21, 24, 26, 29, 31, 34, 36, 39, 41, 44, 46, 49, and 51 wherein an angiogenesis-related transcript is detected where hybridization is detected.
- the polynucleotide may comprise a sequence according to any one of SEQ ID NO:s 2, 4, 7, 9, 12, 14, 17, 19, 21, 24, 26, 29, 31, 34, 36, 39, 41, 44, 46, 49, and 51.
- the biological sample may be a tissue sample, or sample of isolated nucleic acids such as mRNA.
- the nucleic acids may be amplified prior to contacting the biological sample with the polynucleotide. Further, the polynucleotide is immobilized on a solid surface.
- a method of affecting at least one bioactivity selected from angiogenesis and vasculogenesis in a vertebrate organism comprises the step of administering an effective angiogenesis or vasculogenesis affecting amount of a nucleotide or polypeptide described herein to the organism.
- the organism may be mammal, such as mice, rats, rabbits, guinea pigs, cats, dogs, pigs, cows, monkeys, and humans.
- Vasculogenesis or angiogenesis may be enhanced, increased, inhibited, or decreased. This method may be used on organisms that have an angiogenesis-related disorder, such as those disorders described above.
- a transgenic increased angiogenesis laboratory animal which comprises one or more cells in which the expression of a sequence according to any one of SEQ ID NO:s 2, 4, 7, 9, 12, 14, 17, 19, 21, 24, 26, 29, 31, 34, 36, 39, 41, 44, 46, 49, and 51 is upregulated.
- Transgenic decreased angiogenesis laboratory animals are also provided, which comprise one or more cells in which the expression of a sequence according to any one of SEQ ID NO:s 2, 4, 7, 9, 12, 14, 17, 19, 21, 24, 26, 29, 31, 34, 36, 39, 41, 44, 46, 49, and 51 is down regulated or absent.
- angiogenesis-related condition or "angiogenesis-related disease (state)” as used herein mean a condition which is marked by either an excess or a deficit of vessel development or which is improved by an increase or decrease in vessel development.
- Disorders associated with increased angiogenesis include, but are not limited to, cancer (including solid tumors, leukemias, and tumor metastases), benign tumors (including hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas), retinopathy, macular degeneration, and corneal ulceration.
- Pathological states linked to decreased angiogenesis or states which can improve with increased angiogenesis include, but are not limited to, ischemic heart disease, infertility, ulcers, scleradoma, (insufficient) wound healing, ischemia, myocardial infarction, myocardosis, angina pectoris, unstable angina, coronary arteriosclerosis, arteriosclerosis obliterans (ASO), Berger's disease, arterial embolism, arterial thrombosis, cerebrovascular occlusion, cerebral infarction, cerebral thrombosis, cerebral embolism, and stroke.
- ischemic heart disease infertility, ulcers, scleradoma
- (insufficient) wound healing ischemia, myocardial infarction, myocardosis, angina pectoris, unstable angina, coronary arteriosclerosis, arteriosclerosis obliterans (ASO), Berger's disease, arterial embolism, arterial thrombosis, cerebrovascular occlusion,
- angiogenesis related diseases include, but are not limited to, diseases associated with rubeosis (neovasculariation of the angle) and diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue including all forms of prohferative vitreoretinopathy, whether or not associated with diabetes, diseases with symptoms of chronic inflammation, such as inflammatory bowel disease, psoriasis, sarcoidosis and rheumatoid arthritis.
- a "host cell” is a naturally occurring cell or a transformed cell that contains an expression vector and supports the replication or expression of the expression vector.
- Host cells may be cultured cells, explants, cells in vivo, and the like.
- Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa, and the like (see, e.g., the American Type Culture Collection catalog or web site, www.atcc.org).
- polypeptide peptide
- protein protein
- amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
- amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
- Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine.
- Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
- Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
- Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
- nucleic acid sequences conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
- nucleic acid variations are "silent variations," which are one species of conservatively modified variations.
- Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
- each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
- TGG which is ordinarily the only codon for tryptophan
- amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
- label refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
- labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes, biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.
- vector refers to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
- the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
- the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
- stringent hybridization conditions refers to conditions under which sequences will hybridize. Stringent conditions are sequence-dependent and will be different in different circumstances. Skilled workers have access to significant amounts of descriptive material detailing reaction conditions that are appropriate for a given sequence.
- stringent hybridization conditions refers to conditions under which sequences will hybridize. Stringent conditions are sequence-dependent and will be different in different circumstances. Skilled workers have access to significant amounts of descriptive material detailing reaction conditions that are appropriate for a given sequence.
- inhibitors As used herein, the terms “inhibitors,” “activators,” and “modulators” of angiogenic polynucleotide and polypeptide sequences and angiogeneic activity refer to inhibitory, activating, or modulating molecules.
- “Inhibitors” are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of angiogenesis proteins, e.g., antagonists.
- Activators are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate angiogenesis protein activity.
- Inhibitors, activators, or modulators include genetically modified versions of angiogenesis proteins, e.g., versions with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, antibodies, small chemical molecules and the like.
- Assays for inhibitors and activators include, e.g., expressing the angiogenic protein in vitro, in cells, or cell membranes, applying putative modulator compounds, and then determining the functional effects on activity, as described above.
- Figure 1 shows a ratio-ratio plot with the log 2 expression ratio of genes in a cDNA library when compared to embryonic brain endothelial cell portion versus embryonic brain left over portion and adult brain endothelial cell portion versus adult brain left over portion;
- Figure 2 shows a ratio-intensity plot with average intensity versus log 2 expression ratio of genes in a cDNA library when compared to embryonic brain, heart, and skin endothelial cell versus left over embryonic portions and all adult endothelial cells and left over portions;
- Figure 3 schematically depicts microarray data for gene OJC8009J7;
- Figure 4 shows a wild type zebrafish embryo at 28 hpf;
- Figure 5 shows a OJC8009J7 morphant embryo at 28 hpf
- Figure 6 shows a wild type zebrafish embryo at 56 hpf
- Figure 7 shows a OJC8009J7 morphant embryo at 56 hpf
- Figure 8 shows a wild type zebrafish at 48-56 hpf
- Figure 9 shows a OJC8009J7 morphant embryo at 48-56 hpf
- Figure 10 schematically depicts microarray data for gene HUP8001K17
- Figure 11 shows a HUP8001K17 morphant embryo at 28 hpf
- Figure 12 shows a HUP8001K17 morphant embryo at 56 hpf
- Figure 13 shows a HUP8001K17 morphant embryo at 48-56 hpf
- Figure 14 schematically depicts microarray data for gene HUP8001K21
- Figure 15 shows a HUP8001K21 morphant embryo at 28 hpf
- Figure 16 shows a HUP8001K21 morphant embryo at 56 hpf
- Figure 17 shows a HUP8001K21 morphant embryo at 48-56 hpf;
- Figure 18 schematically depicts microarray data for gene HUP8003D24;
- Figure 19 shows a HUP8003D24 morphant embryo at 48-56 hpf
- Figure 20 schematically depicts microarray data for gene HUP8004N1
- Figure 21 shows a HUP8004N1 morphant embryo at 28 hpf
- Figure 22 shows a HUP8004N1 morphant embryo at 56 hpf
- Figure 23 shows a HUP8004N1 morphant embryo at 48-56 hpf
- Figure 24 schematically depicts microarray data for gene HUP8010A10
- Figure 25 shows a HUP8010A10 morphant embryo at 28 hpf
- Figure 26 shows a HUP8010A10 morphant embryo at 56 hpf
- Figure 27 shows a HUP8010A10 morphant embryo at 48-56 hpf;
- Figure 28 schematically depicts microarray data for gene NOC8003L17;
- Figure 29 shows aNOC8003L17 morphant embryo at 28 hpf
- Figure 30 shows aNOC8003L17 morphant embryo at 56 hpf
- Figure 31 shows aNOC8003L17 morphant embryo at 48-56 hpf
- Figure 32 schematically depicts microarray data for gene NOC8009C9
- Figure 33 shows a NOC8009C9 morphant embryo at 28 hpf
- Figure 34 shows a NOC8009C9 morphant embryo at 56 hpf
- Figure 35 shows aNOC8009C9 morphant embryo at 48-56 hpf
- Figure 36 schematically depicts microarray data for gene NOC8009G23
- Figure 37 shows aNOC8009G23 morphant embryo at 28 hpf;
- Figure 38 shows a NOC8009G23 morphant embryo at 56 hpf;
- Figure 39 shows aNOC8009G23 morphant embryo at 48-56 hpf
- Figure 40 schematically depicts microarray data for gene OJC8003C9
- Figure 41 shows a OJC8003C9 morphant embryo at 28 hpf
- Figure 42 shows a OJC8003C9 morphant embryo at 56 hpf; and Figure 43 shows a OJC8003C9 morphant embryo at 48-56 hpf.
- a cDNA Library was prepared by collecting the mRNA from purely isolated adult and embryonic mice vascular fragments. The collected mRNA was used to develop cDNA libraries with a broad coverage of genes expressed in the vasculature. Because of the variety in mouse age, the vascular genes represented those active at different times and in different situations in the vasculature.
- microarrays were created by printing DNA from the cDNA library onto a solid support as known in the art. The microarrays were used to reveal the gene candidates through gene expression profiling. Select tissues from adult and E 18.5 embryonic mice were collected. Tissue selection was based on the amount and purity of RNA available for extraction. After tissues were removed, they were separated into two portions using antibodies or lectins. The first portion, the endothelial cell fraction or EC, contained endothelial cells as well as pericytes and vascular smooth muscle cells which are tightly associated with the vascular fragments. The second portion, also referred to as the left over portion or LO, were those cells remaining after the EC was isolated.
- the first portion the endothelial cell fraction or EC
- the second portion also referred to as the left over portion or LO, were those cells remaining after the EC was isolated.
- RNA from adult mice brain and heart tissues were used, both the EC and LO of each. From embryonic mice, brain, heart, and skin, both EC and LO of each, were utilized. The RNA from each fraction was extracted. A common reference RNA (Universal Mouse Reference RNA; Stratagene, Inc.) was employed at this stage for reference purposes. The isolated RNA and the reference RNA were reverse transcribed then amplified twice through two rounds of antisense RNA amplification. The isolated RNA was labelled with the fluorophore cyanine-3 and the reference RNA with cyanine-5.
- RNA was assayed through hybridization with the microarrays described above.
- the hybridized microarrays were scanned and image analysis used to process the experimental data. Normalizing the data through a signal intensity-based normalization algorithm allowed for statistical evaluation of differentially expressed genes. Genes exhibiting differential expression were selected for further analysis.
- Figure 1 shows a ratio-ratio plot of the data values obtained through comparisons between embryonic brain EC and LO genes and between adult brain EC and LO genes. A comparison of total adult EC with total embryonic EC was also conducted, data not shown. Data points represented with 'DTI candidates' or 'DT2 candidates' were generally upregulated (>0 log expression ratio).
- a total of ten genes of interest were selected for further analysis. Specific expression data for each gene follows throughout and includes a graph showing that gene's expression profile. Tables are used to show the intensity of the microarray signal, the log 2 expression ratio, p-value, and rank (rank given only for certain fields). The highest rank was awarded to the gene with the highest expression ratio value, the lowest rank was assigned to the gene with the lowest expression value, based on expression ratio values. P-values are given as a value from zero to one. Nalues close to one indicate a gene that is upregulated, whereas- values close to zero indicate a statistically down regulated gene. A statistically significant p-value of 0.05 corresponds to a p-value of 0.05 or 0.95.
- eec/r refers to embryonic EC portions versus all remaining portions
- ec/lo refers to all EC portions versus all LO portions
- abeclo refers to adult brain EC portion versus adult brain LO portion
- abecebec refers to adult brain EC portion versus embryonic brain EC portion
- aheclo refers to adult heart EC portion versus adult heart LO portion
- ebeclo refers to embryonic brain EC portion versus embryonic brain LO portion
- eheclo refers to embryonic heart EC portion versus embryonic heart LO portion
- eseclo refers to embryonic skin EC portion versus embryonic skin LO portion.
- the process involves the use of specific antisense oligonucleotides that block translation from targeted mR A molecule(s). This allows for inhibition of the gene of interest and allows for a determination of gene function in the development and health of the zebrafish.
- Zebrafish share genes for vertebrate functions with mammalian vertebrates such as mice and humans. Studies have demonstrated that organ and/or tissue development in zebrafish can reliably predict effects in humans (See, ter alia, Shin and Fishman, From zebrafish to humans: Modular medical models, Ann. Rev. Genomics and Human Genet.
- the zebrafish homolog of the target gene was identified. Then, a specific morpholino phosphorodiamidate oligonucleotide was designed to match the AUG initiation codon or splice acceptor/donor site of the target gene. To create a stock solution of morpholino, pellets containing 100 nmoles of the phosphorodiamidate oligonucleotides were dissolved in 33.3 ⁇ l milli-Q water, giving a concentration of 25 mg/ml, and stored at -20°C.
- injection solution 8 ⁇ l of the stock solution was added to 92 ⁇ l of sterile-filtered IX Danieu buffer (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO 4 , 0.6 mM Ca(NO 3 ) 2 , 5 mM HEPES, pH 7.6) supplemented with 15mM Tris-Cl, pH 8.0.
- the 2 mg/ml injection solution was also stored at -20°C.
- the materials and embryos were maintained at approximately 28°C. Injection needles were calibrated so that injection times could optimally be within a range of 100-600 msec. Embryos from the one cell stage to the early eight cell stage were used. The morpholinos were microinjected into the yolk sac. Specific injection volumes, or effective dose of the morpholino, are described below. Typical initial doses included 3, 6, and 12 ng (1.5, 3, and 6 nl, respectively). Toxicity at the 3 nl dose resulted in subsequent doses of 0.5, 1, and 2 ng (1, 2, and 4 nl, respectively). Approximately 40 embryos were injected at each dose level, and approximately 40 embryos were retained as non-injected controls.
- the embryos engineered to have a knockdown of the specific gene were allowed to develop (See Nasevicius and Ekker, Effective targeted gene 'knockdown' in zebrafish, Nature Genetics vol 26, October 2000.). The embryos were monitored throughout development, both by examining morphology and undertaking specific analysis and assays of developing tissues.
- double morpholino injections were performed as well. Specific injection volumes for double injections are described below.
- PTU propyl thioracil
- microangiopathy and in situ hybridization with fli-1, flk-1, flt-4, tie-1, tie-2, and cdh5 were conducted. At least 120 embryos were administered the double morpholino dose, of which at least 100 were harvested at 24 hpf for in situ hybridization with the above-noted molecular markers. Remaining embryos were used for microangiopathy. Data specific to the evaluation of each of the ten targets are described below.
- the morphology observations conducted at 24-28 hpf included an indication of whether the embryos exhibited general delay relative to control embryos. Further, cell death type and degree were recorded, general embryo shape and brain morphology were recorded as well. Finally, yolk sac edema, if present, was evaluated and recorded, as was heart morphology.
- double morpholino embryos were evaluated for in situ hybridization of fli-1, flk-1, flt-4, tie-1, tie-2, and cdh5.
- Those embryos showing a loss of 1-35% of intersegmental expression were considered to have a low effect
- those embryos showing a loss of 36-70% of intersegmental expression were considered to have a medium effect
- those embryos showing a loss of 71-100% of intersegmental expression were considered to have a high effect.
- Microangiopathy was also evaluated at 48hpf in double morpholino embryos.
- the embryos were transferred into a tricaine solution and the sinus venosa/common cardinal vein was injected with 10 ⁇ l FITC-Dextran solution (2,000,000 Da, 20 mg/ml).
- SEQ ID NO:l The gene having the sequence shown in SEQ ID NO:l was identified as selectively expressed in blood vessels based on microarray data, see Figure 3. Specific data are given below in Table 1. Using sequence and annotation databases the equivalent gene in mice (SEQ ID NO:2) and the human homolog (SEQ ID NO:4) was also deduced. Proteins encoded by these sequences are given at SEQ ID NO:s 3 and 5, respectively. Table 1: Expression profile data for gene OJC8009J7
- the gene was further analyzed in zebrafish embryos.
- One corresponding zebrafish gene was identified for targeting.
- Two morpholinos were prepared, szl75 and szl76, each targeted to the zebrafish gene.
- Two (2)ng of szl75 morpholino and 12ng of szl76 morpholino were administered to each fertilized egg. The embryos were allowed to develop.
- a secondary in situ hybridization screen with six different probes was conducted.
- the probe fli-1 revealed that 7% of the 14 morphant embryos assayed had low effects, and 29% had high effects, that is, loss of 71-100% of intersegmental expression.
- the probe flk-1, NEGF receptor 2 indicated that 25% of the 12 morphants reviewed had low effects and 8% had high effects.
- the probe tie-1 indicated that 11% of the 9 morphants observed had medium effects and 11% had high effects.
- the probe flt-4, NEGF receptor 3 indicated that all 10 morphants observed were normal, and the probe tie-2 indicated that all 6 morphants observed were normal.
- Table 2 secondary in situ hybridization data At 28 hpf embryos were observed morphologically.
- Figure 5 shows a representative morphant embryo at 28 hpf. As evidenced from the figure, particularly when viewed in light of the 28 hpf wild type embryo of Figure 4, the morphants exhibited normal morphology.
- Figure 7 a representative morphant embryo is shown in Figure 7. The normal morphology observed in the embryos can be easily understood when Figure 7 is viewed in light of the 56 hpf wild type embryo of Figure 6.
- SEQ ID NO:6 The gene having the sequence shown in SEQ ID NO:6 was identified as selectively expressed in blood vessels based on microarray data, see Figure 10. Specific data are given below in Table 3. Using sequence and annotation databases the equivalent gene in mice (SEQ ID NO:7) and the human homolog (SEQ ID NO:9) was also deduced. Proteins encoded by these sequences are given at SEQ ID NO:s 8 and 10, respectively.
- Table 3 Expression profile data for gene HUP8001K17.
- the gene was further analyzed in zebrafish embryos.
- One corresponding zebrafish gene was identified for targeting.
- Two different morpholinos were prepared, szl43 and szl44, each targeted to the zebrafish gene.
- Different amounts of morpholinos were administered as described below.
- the predetermined amount of each morpholino was administered to each fertilized egg.
- the embryos were allowed to develop.
- 24 hpf secondary in situ hybridization screens with six different probes were conducted.
- Four probes specifically selected to analyze axial and intersegmental vessel expression revealed the following: using the fli-1 probe, 6% of the 17 embryos analyzed had medium intersegmental expression effects. Another 18% had high effects.
- the probe flk-1, NEGF receptor 2 indicated that 20% of the 15 morphants reviewed had medium and 13% had high effects. When analyzed through the probe tie-1, 63% of the 16 morphants observed had high effects.
- the probe flt-4, NEGF receptor 3 indicated that all 14 morphants observed were normal, and the probe tie-2 showed all 18 observed morphants as normal.
- the other screen was performed on embryos that received 1.5 ng of szl43 morpholino and 6 ng of szl44 morpholino.
- the probe fli-1 indicated that 42% of the 12 morphants analyzed had high effects.
- the flk-1 probe demonstrated that 23%of the 13 morphants observed had high effects.
- the tie-1 probe revealed 54% of 13 morphants had high effects.
- the probe cdh5 indicated that 15% of 27 morphants had medium effects and another 11% had high effects.
- the probe flt-4 indicated that all 14 morphants observed were normal. And the probe tie-2, showed all 18 observed morphants as normal.
- FIG. 1 shows a representative embryo at 28 hpf.
- the morphant embryos received a 1.5 ng dose of szl43 and a 6 ng dose of szl44, all did not exhibit normal morphology.
- a representative embryo is shown in Figure 11. Twenty embryos were observed, 50% of them showed a curly down body, indicated by the arrowhead in Figure 11, with yolk tube extension, indicated with a short arrow. Mild cell death was observed in 60% of the embryos, as shown by the long arrow in Figure 11. Finally, 50% of the embryos had yolk cell edema.
- the arrow points to an area of reduced intersegmental vasculature, and the arrowhead indicates a point of leaky vasculature.
- Figure 8 a wild type embryo at this time stage is shown in Figure 8. The experimental data reveal that the gene is expressed by scattered cells in many organs, but most clearly seen in the CNS.
- Gene HUP8001K21 The gene having the sequence shown in SEQ ID NO: 11 was identified as selectively expressed during angiogenesis based on microarray data, see Figure 14. Specific data are given below in Table 5. Using sequence and annotation databases the equivalent gene in mice (SEQ ID NO: 12) and the human homolog (SEQ ID NO: 14) was also deduced. Proteins encoded by these sequences are given at SEQ ID NO:s 13 and 15, respectively.
- sz257 morpholino 12 ng of sz258 morpholino were administered to each fertilized egg. The embryos were allowed to develop. At 24 hpf a secondary in situ hybridization screen with six different probes was conducted.
- Figure 15 shows a representative morphant embryo at 28 hpf.
- yolk sac edema was observed in 47% of the 55 morphants analyzed.
- a representative morphant embryo is shown in Figure 16.
- expanded hindbrain was found in 34% of embryos.
- Yolk sac edema shown by a short arrow, was also observed in 58% of embryos.
- An arrowhead points out the location checked for pericardial edema; it was not observed.
- SEQ ID NO: 16 The gene having the sequence shown in SEQ ID NO: 16 was identified as selectively expressed during angiogenesis based on microarray data, see Figure 18. Specific data are given below in Table 7. Using sequence and annotation databases the equivalent gene in mice (SEQ ID NO: 17) and two human homologs (SEQ ID NO:sl9 and 21) was also deduced. Proteins encoded by these sequences are given at SEQ ID NO:s 18, 20, and 22, respectively. Table 7: Expression profile data for gene HUP8003D24
- the gene was further analyzed in zebrafish embryos. Three corresponding zebrafish genes were identified for targeting. Two morpholinos were prepared, szl85 and szl86, which were targeted to the three zebrafish genes. For the lower dose group, 3ng of szl85 morpholino and 6ng of szl86 morpholino were administered to each fertilized egg. For the double dose group, 6ng of szl85 morpholino and 12ng of szl86 morpholino were administered. The embryos were allowed to develop. At 24 hpf a secondary in situ hybridization screen with six different probes was conducted.
- Intersegmental expression was analyzed in the assay and results differed somewhat based on the probe used.
- the probe fli-1 revealed that 18% of the 11 morphant embryos assayed had medium effects, i.e., 36-70% loss of intersegmental expression, and 18% had high effects.
- the probe flk-1 indicated that 36% of the 11 morphants reviewed had high effects.
- the probe tz ' e-i indicated that 29% of the 14 morphants observed had high effects.
- the probe cdh5 indicated 31% of the 16 morphants observed had high effects, and 31% had medium effects.
- the probe flt-4 indicated that all 16 morphants observed were normal, and the probe tie- 2 indicated that all 15 morphants observed were normal.
- the probe fli-1 revealed that 33% of the 3 morphant embryos assayed had low effects and 33% had medium effects.
- the probe flk-1 indicated that 100%, or both of the 2 morphants reviewed, had medium effects.
- the probe cdh5 indicated 100%, all 7 of the morphants observed had high effects.
- the probe flt-4 indicated that all 3 morphants observed were normal, and the probe tie-2 indicated that all 7 morphants observed were normal.
- Table 8 summarizes the foregoing data.
- Microangiography on morphants given the lower dose of morpholinos (3ng szl85, 6ng szl86) was used to locate the presence of FITC-Dextran in various regions of the embryo, see Figure 19. Of the 33 embryos, none had FITC-Dextran in the heart, or the heart and head. Reduced intersegmental vasculature was seen as indicated by the arrow in Figure 19. High effects were observed in 6% of the embryos, medium effects in 15% and low effects in 36%. No leaky vasculature was observed. Normal embryos accounted for 43% of the sample. The data reveal that the gene is expressed in many locations, such as vessels and epithelial structures in the kidneys as well as in large vessels, megakaryocytes, in heart valves and in the skin epithelium.
- SEQ ID NO:23 The gene having the sequence shown in SEQ ID NO:23 was identified as selectively expressed during angiogenesis based on microarray data, see Figure 20. Specific data are given below in Table 9. Using sequence and annotation databases the equivalent gene in mice (SEQ ID NO:24) and the human homolog (SEQ ID NO:26) was also deduced. Proteins encoded by these sequences are given at SEQ ID NO:s 25 and 27, respectively.
- the gene was further analyzed in zebrafish embryos. Two corresponding zebrafish genes were identified for targeting. Two morpholinos were prepared, sz223 and sz224, each targeted to one of the zebrafish genes. Two dosing strategies were employed. The first dose group received 2ng of sz223 morpholino and Ing of sz224 morpholino in each fertilized egg. The second dose group received Ing of sz223 morpholino and 0.5ng of sz224 morpholino in each fertilized egg. The embryos were allowed to develop. At 24 hpf a secondary in situ hybridization screen with six different probes was conducted.
- Figure 21 shows a representative morphant embryo at 28 hpf.
- yolk sac edema was observed in 56% of the 59 morphants studied.
- a representative morphant embryo is shown in Figure22.
- pericardial edema was observed in 35% of embryos, 65% had a blood pool in the yolk, also indicated by the arrow, and 30% showed reduced IS blood flow.
- SEQ ID NO:28 The gene having the sequence shown in SEQ ID NO:28 was identified as selectively expressed during angiogenesis based on microarray data, see Figure 24. Specific data are given below in Table 11. Using sequence and annotation databases the equivalent gene in mice (SEQ ID NO:29) and the human homolog (SEQ ID NO:31) was also deduced. Proteins encoded by these sequences are given at SEQ ID NO:s 30 and 32, respectively.
- Table 11 Expression profile data for gene HUP8010A10
- the gene was further analyzed in zebrafish embryos.
- a corresponding zebrafish genes was identified for targeting.
- Two morpholinos were prepared, sz267 and sz268, each targeted to one of the zebrafish genes.
- 4ng of sz267 morpholino and 2ng of sz268 morpholino were administered to each fertilized egg.
- 6ng of sz267 morpholino and 3ng of sz268 morpholino were administered.
- the embryos were allowed to develop.
- a secondary in situ hybridization screen with six different probes was conducted.
- the probe fli-1 revealed that 25% of the 16 morphant embryos assayed had low effects, and 19% had high effects.
- the probe flk-1 indicated that 33% of the 6 morphants reviewed had low effects.
- the probe tie-1 indicated that 67% of the 15 morphants observed had high effects.
- the probe cdh5 indicated that 21% of the 24 morphants observed had low effects, 13% had medium effects, and 29% had high effects.
- the probe flt-4 indicated that all 15 morphants observed were normal, and the probe tie-2 indicated that all 15 morphants observed were normal.
- Table 12 summarizes the foregoing data.
- Figure 25 shows a representative morphant embryo at 28 hpf.
- Cell death in the head was observed in 47% of the 61 embryos observed, as indicated by the arrow in Figure 25.
- the arrowhead indicates expanded hindbrain, which was seen in 51% of embryos. Mild yolk sac edema was observed in 21% of the morphants.
- Figure 26 a representative morphant embryo is shown in Figure 26.
- the arrow indicates expanded hindbrain, which was seen in 44% of embryos. Mild yolk sac edema was observed in 29% of the morphants and is indicated by the arrowhead.
- Reduced IS blood flow was noted in 14% of the embryos, and reduced blood flow was found in 17% of the embryos.
- Microangiography on morphants was used to locate the presence of FITC-Dextran in various regions of the embryo, see Figure 27. Of the 32 embryos, none had FITC-Dextran in the heart, or the heart and head. Reduced intersegmental vasculature was seen as indicated by the arrow in Figure 27. High effects were observed in 6% of the embryos, medium effects in 3% and low effects in 34%. No leaky vasculature was observed. Normal embryos accounted for 56% of the sample. Gene NOC8003L17
- SEQ ID NO:33 The gene having the sequence shown in SEQ ID NO:33 was identified as selectively expressed in blood vessels based on microarray data, see Figure 28. Specific data are given below in Table 13. Using sequence and annotation databases the equivalent gene in mice (SEQ ID NO:34) and the human homolog (SEQ ID NO:36) was also deduced. Proteins encoded by these sequences are given at SEQ ID NO:s 35 and 37, respectively.
- the gene was further analyzed in zebrafish embryos.
- One corresponding zebrafish gene was identified for targeting.
- Two morpholinos were prepared, szl80 and szl81, each targeted to the zebrafish gene.
- 12ng of szl80 and Ing of szl ⁇ l were administered to each fertilized egg.
- 12ng of szl80 and 2ng of szl81 were administered to each fertilized egg.
- the embryos were allowed to develop.
- a secondary in situ hybridization screen with six different probes was conducted.
- Figure 29 shows a representative morphant embryo. As indicated by the arrow, yolk sac edema was observed in 67% of the 48 morphants studied. Embryos observed at 56 hpf demonstrated normal morphology, a representative embryo is shown in Figure 30.
- SEQ ID NO:38 The gene having the sequence shown in SEQ ID NO:38 was identified as selectively expressed during angiogenesis based on microarray data, see Figure 32. Specific data are given below in Table 15. Using sequence and annotation databases the equivalent gene in mice (SEQ ID NO:39) and the human homolog (SEQ ID NO:41) was also deduced. Proteins encoded by these sequences are given at SEQ ID NO:s 40 and 42, respectively. Table 15: Expression profile data for gene NOC8009C9
- the gene was further analyzed in zebrafish embryos.
- One corresponding zebrafish genes was identified for targeting.
- Two morpholinos were prepared, sz241 Three (3)ng of sz241 morpholino and Ing of sz242 morpholino were administered to each fertilized egg. The embryos were allowed to develop.
- a secondary in situ hybridization screen with six different probes was conducted.
- the probe fli-1 revealed that 7% of the 15 morphant embryos assayed had medium effects.
- the probe flk-1 indicated that all 15 morphants observed were normal.
- the probe tie-1 indicated that 7% of the 15 morphants observed had high effects.
- the probe cdh5 indicated that 15% of the 20 morphants observed had low effects.
- Medium effects were seen in 5% and high effects in 15%.
- the probe flt-4 indicated that all 15 morphants observed were normal, and the probe tie-2 indicated that all 16 morphants observed were normal.
- Figure 33 shows a representative morphant embryo at 28 hpf. Mild cell death was observed in 25% of the 53 embryos observed. At 56 hpf 52 embryos were observed for phenotypic characteristics, a representative morphant embryo is shown in Figure 34. As indicated by the arrow, pericardial edema was seen in 13% of embryos. The arrowhead points toward a region of yolk sac edema, seen in 25% of embryos. Reduced IS blood flow was observed in 13% and 15% showed reduced axial blood flow.
- SEQ ID NO:43 The gene having the sequence shown in SEQ ID NO:43 was identified as selectively expressed during angiogenesis based on microarray data, see Figure 36. Specific data are given below in Table 17. Using sequence and annotation databases the equivalent gene in mice (SEQ ID NO:44) and the human homolog (SEQ ID NO:46) was also deduced. Proteins encoded by these sequences are given at SEQ ID NO:s 45 and 47, respectively.
- the gene was further analyzed in zebrafish embryos.
- One corresponding zebrafish gene was identified for targeting.
- Two morpholinos were prepared, szl49 and szl50, each targeted to the zebrafish gene.
- 1.5ng of szl49 morpholino and 1.5ng of szl50 morpholino were administered to fertilized eggs.
- 2ng of szl49 morpholino and 2ng of szl50 morpholino were administered to fertilized eggs.
- 3ng of szl49 morpholino and 3ng of szl50 morpholino were administered to fertilized eggs.
- the embryos were allowed to develop.
- a secondary in situ hybridization screen with six different probes was conducted.
- the probe fli-1 revealed that 13% of the 15 morphant embryos assayed had medium effects, 27% had high effects.
- the probe flk-1 indicated that 7% of the 15 morphants reviewed had low effects, 7% had medium effects and 20% had high effects.
- the probe tie-1 indicated that 62% of the 13 morphants observed had high effects.
- the probe cdh5 indicated that 25% of the 12 morphants observed had high effects, some with breaks in axial expression.
- the probe flt-4 indicated that 13% of the 15 morphants observed had low effects in the axial vessels, including breaks in axial expression and severely malformed tails.
- the probe tie-2 indicated that all 8 morphants observed were normal.
- Figure 37 shows a representative morphant embryo at 28 hpf.
- cell death was observed in 40% of the 20 embryos observed.
- yolk sac edema was observed in 55% of the morphants.
- Curly down body was seen in 40% of morphants.
- Figure38 a representative morphant embryo is shown in Figure38.
- pericardial edema was observed in 55% of morphants.
- the arrowhead points toward yolk sac edema, observed in 55% of morphants. Curly down body was reported in 30% of embryos.
- a primary in situ hybridization screen with cdh5 on 22 morphants from the first dose group showed 90% as normal. Low effects of reduced intersegmental expression and curly down embryos were seen in 5% of the embryos, and high effects with very short tails were seen in 5% of embryos.
- a primary in situ hybridization screen with cdh5 was also performed on 20 morphants from the third dose group, showing 80% as normal. Low effects of reduced intersegmental expression were seen in 5% of the embryos, and medium effects in 15% of embryos.
- Microangiography on morphants was used to locate the presence of FITC-Dextran in various regions of the embryo, see Figure 39. Of the 25 embryos, none had FITC-Dextran in the heart, or the heart and head. Reduced intersegmental vasculature was seen in 24% of the embryos as indicated by the arrow in Figure 39. No leaky vasculature was observed. Normal embryos accounted for 76% of the sample, in example of normal intersegmental vessels is indicated by the arrowhead.
- SEQ ID NO:48 The gene having the sequence shown in SEQ ID NO:48 was identified as selectively expressed in blood vessels based on microarray data, see Figure 40. Specific data are given below in Table 19. Using sequence and annotation databases the equivalent gene in mice (SEQ ID NO:49) and the human homolog (SEQ ID NO:51) was also deduced. Proteins encoded by these sequences are given at SEQ ID NO:s 50 and 52, respectively. Table 19: Expression profile data for gene OJC8003C9
- One corresponding zebrafish gene was identified for targeting.
- Two morpholinos were prepared, szl29 and szl30, each targeted to the zebrafish gene.
- 3ng of szl29 morpholino and 4.5ng of szl30 morpholino were administered to each fertilized egg.
- 4ng of szl29 morpholino and 6ng of szl30 morpholino were administered to each fertilized egg.
- 6ng of szl29 morpholino and 8ng of szl30 morpholino were administered to each fertilized egg.
- szl29 morpholino 6ng of szl29 morpholino and 9ng of szl30 morpholino were administered to each fertilized egg. The embryos were allowed to develop. At 24 hpf a secondary in situ hybridization screen with six different probes was conducted.
- the probe fli-1 revealed that 10% of the 10 morphant embryos assayed had medium effects, and 50% had high effects.
- the probe flk-1 indicated that 25% of the 12 morphants reviewed had medium effects and 25% had high effects.
- the probe tie-1 indicated that 17% of the 6 morphants observed had low effects, and 50% had high effects.
- the probe cdh5 indicated that 40% of 5 morphants observed had medium effects.
- the probe flt- 4 indicated that all 9 morphants observed were normal, and the probe tze-2 indicated that all 6morphants observed were normal.
- Table 20 summarizes the foregoing data.
- Figure 41 shows a representative morphant embryo at 28 hpf. As indicated by the arrow, curly down body was found in 35% of the 20 morphants observed. At 56 hpf a total of 20 embryos were observed for phenotypic characteristics, a representative morphant embryo is shown in Figure 42. As indicated by the long arrow, 60% of the embryos had cell death with an associated expanded hindbrain ventricle. Yolk sac edema was observed in 25% of embryos, as indicated by the short arrow. The arrowhead points out the lack of pericardial edema associated with the yolk sac edema.
- the present invention relates to the ten gene targets, and proteins related thereto, which were originally identified as upregulated during vasculogenesis or angiogenesis through microarray evaluation and subsequently proven to play a critical role in vivo with zebrafish embryo experimentation.
- genes and proteins can form the basis of novel methods and treatments directed to angiogenesis-related conditions. For example, biological samples from a patient suspected of suffering from an angiogenesis-related condition can be screened to ascertain if genes or proteins of the present invention are expressed at the correct time, location, and intensity in the patient. Such screening methods form part of the claimed invention. If a gene and/or protein is identified as improperly expressed, therapies to correct the condition such as gene therapy or medicament can be initiated according to methods and procedures described herein or known in the art. With such specific data as is now possible using tools described herein, rapid diagnosis and specific, targeted treatment is possible.
- screening method relies on gene amplification for detecting patients with conditions related to vasculogenesis or angiogenesis. Such methods could employ PCR, in situ hybridisation, and/or Southern blotting techniques to elucidate the condition. Another type of screening method could be based on evaluations of gene expression, using known techniques such as quantitative PCR, microarrays, Northern blotting, or in situ hybridisation. Yet another type of screening method that could be used would measure or monitor protein expression and could be effected with techniques such as immunohistochemistry, Western blotting, ELISA, or FACS.
- an angiogenesis-related condition could be improved through administration of compounds containing genes and/or proteins according to the invention, one or more of the genes and/or proteins could be administered together or sequentially by methods known in the art.
- Isolated nucleic acid molecules or proteins of the present invention can be obtained, for example, by synthesis using standard direct peptide synthesizing techniques or recombinant methods. Proteins may be isolated or purified in a variety of ways known to those skilled in the art, such as electrophoretic purification or chromatographic teclmiques.
- Administration of the compounds of the present invention can be effected by any method that enables delivery of the compounds to the site of desired action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), and topical administration.
- Gene therapy approaches may be used to introduce nucleotides of the present invention into a cell, group of cells, or organism. Both in vivo and ex vivo methods can be utilized.
- Vectors typically are used in this procedure.
- Non-virus or virus vectors could be employed, for example recombinant adenovirus or retrovirus.
- the desired gene is introduced into a DNA virus or RNA virus, such as avirulent retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, SV40, and immunodeficiency virus (HIV).
- the recombinant virus is then infected into the target cell(s).
- Multiple genes could be incorporated in a single vector, alternatively, they could be introduced to the target cell(s) in separate vectors simultaneously or sequentially.
- siRNA comprises a sense and antisense strand of RNA corresponding to the gene of interest, for example, SEQ ID NO:2.
- a siRNA molecule consists of approximately 19 nucleotides plus an overhang of approximately 2 nucleotides at the 3' end. Some preferred methods include between 19-23 nucleotides plus 3' overhang.
- the siRNA is introduced to the cell or cells of interest through known methods. Following introduction, the cell or cells destroy ssRNA having the same sequence. This results in a reduction or prevention in translation of a targeted gene and a corresponding reduction or prevention in protein production.
- the amount of active compound administered can be determined after assessing the subject being treated, the severity of the disorder or condition, the rate of administration, and the disposition of the compound. Doses may be administered all at once, or spread out over a discrete time period.
- compositions may include carriers, adjuvants, buffers, or excipients as known in the art. If desired, the compositions may further contain ingredients such as flavorings, sweeteners, binders, dyes, lubricating agents, perfume, thickening agents, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, and pH regulating agents.
- Compositions may be in any suitable form, for example, tablet, capsule, pill, powder, sustained release formulation, solution, suspension, emulsion, ointment or cream. The compositions may be sterile. Methods of preparing various pharmaceutical compositions with a specific amount of active compound are known or apparent to those skilled in this art.
- the pharmaceutical compositions of the present invention that have been described can be applied to all diseases that require vasculogenic or angiogenic therapy.
- one method for the treatment of an angiogenesis-related disorder involves a composition according to the present invention used to vascularize ischemic tissue.
- a composition according to the present invention used to vascularize ischemic tissue.
- Such methods are well known in the art and include, for example, imaging techniques such as MRI to evaluate myocardial disease.
- the compositions can be administered to increase angiogenesis in tissue affected by or at risk of being affected by a vascular occlusion. This could be an effective means of preventing and/or attenuating ischemia in such tissue.
- Methods are known in the art to evaluate and measure the degree to which ischemia has been attenuated.
- Further treatment methods according to the present invention include the use of any known technique that permits visualization, measurement, and/or evaluation of the functionality and degree of ischemia of the patient's heart. Such evaluations could be made prior to initiating treatment, during the course of treatment, after treatment has been completed, or at some or all stages. Examples of such techniques include echocardiography, cardiovascular nuclear imaging, magnetic resonance imaging, and contrast angiography.
- the present invention takes a step forward in the understanding of vasculogenesis and angiogenesis, and treatments for conditions related to the same, there is still a need in the art to further understand these conditions. Therefore, the present invention further contemplates the creation and use of non-human transgenic animals which could be used for analysis and experimentation. Transgenic animals containing mutant, knock-out or modified genes corresponding to those disclosed herein are therefore also included in the invention. Transgenic animals are genetically modified animals into which recombinant, exogenous or cloned genetic material has been experimentally transferred. Such genetic material is often referred to as a transgene.
- the nucleic acid sequence of the transgene may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene.
- the transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal.
- Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection as known in the art.
- the method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method that favors co- transformation of multiple nucleic acid molecules.
- Detailed procedures for producing transgenic animals are available to one skilled in the art, for example, U.S. Pat. Nos. 5,489,743 and 5,602,307.
- Transgenic technology can be used to produce animals which lack one or more of the ten genes described above.
- knockout animals can be used, especially when their growth and development is measured against data from a wild type or control animal, to elucidate timing and function of the deleted gene(s).
- these animals could also be engineered to exhibit angiogenesis-related disease states, thus furthering the understanding of the role of the particular gene(s) in the progression of the selected disease. This knowledge would be an advance in the state of the art and could lead to promising new therapies for the prevention, management, and cure of disease.
- transgenic animals include replacement of one or more of the above-identified gene(s) in the research organism with the human homolog of the gene.
- a transgenic mouse whose gene corresponding to SEQ ID NO:7 has been replaced with the human homolog, SEQ ID NO:9. While it is accepted that research into effective drug therapies can be conducted in animal models, such a transgenic mouse could be a more effective screening tool into potential drug candidates for human use.
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Abstract
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Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
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US10/581,761 US20090036362A1 (en) | 2003-12-05 | 2004-12-06 | Angiogenesis Affecting Polypeptides, Proteins, and Composition, and Methods of Use Thereof |
NZ547877A NZ547877A (en) | 2003-12-05 | 2004-12-06 | Angiogenesis affecting polypeptides, proteins, and compositions, and methods of use thereof |
CA002548208A CA2548208A1 (en) | 2003-12-05 | 2004-12-06 | Angiogenesis affecting polypeptides, proteins, and compositions, and methods of use thereof |
EP04801722A EP1699818A2 (en) | 2003-12-05 | 2004-12-06 | Angiogenesis affecting polypeptides, proteins, and compositions, and methods of use |
JP2006542541A JP2007512835A (en) | 2003-12-05 | 2004-12-06 | Polypeptides, proteins and compositions affecting angiogenesis and methods of using the same |
AU2004295654A AU2004295654A1 (en) | 2003-12-05 | 2004-12-06 | Angiogenesis affecting polypeptides, proteins, and compositions, and methods of use thereof |
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US48174103P | 2003-12-05 | 2003-12-05 | |
SE0303268A SE0303268D0 (en) | 2003-12-05 | 2003-12-05 | Angiogenesis affecting polypeptides, proteins, and compositions, and methods of use thereof |
SE0303268-7 | 2003-12-05 | ||
US60/481,741 | 2003-12-05 |
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JP (1) | JP2007512835A (en) |
AU (1) | AU2004295654A1 (en) |
CA (1) | CA2548208A1 (en) |
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Cited By (3)
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US20100254946A1 (en) * | 2007-09-25 | 2010-10-07 | Life & Brain Gmbh | Maintenance of hair growth and treatment of hair-loss |
US10730940B2 (en) | 2011-09-23 | 2020-08-04 | Oncomed Pharmaceuticals, Inc. | VEGF/DLL4 binding agents and uses thereof |
US11339213B2 (en) | 2015-09-23 | 2022-05-24 | Mereo Biopharma 5, Inc. | Methods and compositions for treatment of cancer |
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EP1114862A3 (en) * | 1999-11-17 | 2003-08-06 | Switch Biotech Aktiengesellschaft | Use of polyeptides or their encoding nucleic acids for the diagnosis or treatment of skin diseases and their use in indentifying pharmacologically acitve substances |
US7250495B2 (en) * | 2001-06-20 | 2007-07-31 | Genentech, Inc. | PRO20044 polypeptides |
US20030159168A1 (en) * | 2001-09-24 | 2003-08-21 | Wisotzkey Robert G. | CTSZ disruptions, compositions and methods relating thereto |
AU2002367732A1 (en) * | 2001-10-31 | 2003-09-09 | Children's Hospital Medical Center | Method for diagnosis and treatment of rheumatoid arthritis |
JP2005510251A (en) * | 2001-11-29 | 2005-04-21 | ノバルティス アクチエンゲゼルシャフト | Evaluation method and prognostication method of sarcoidosis |
AU2003220387A1 (en) * | 2002-03-19 | 2003-10-08 | Tularik Inc. | Gene amplification in cancer |
EP1347059A1 (en) * | 2002-03-20 | 2003-09-24 | Biofrontera Pharmaceuticals AG | Cathepsin Y for the development of a medicament for the treatment of stroke |
EP1594447A2 (en) * | 2002-10-02 | 2005-11-16 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of tumor |
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Cited By (4)
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US20100254946A1 (en) * | 2007-09-25 | 2010-10-07 | Life & Brain Gmbh | Maintenance of hair growth and treatment of hair-loss |
US10730940B2 (en) | 2011-09-23 | 2020-08-04 | Oncomed Pharmaceuticals, Inc. | VEGF/DLL4 binding agents and uses thereof |
US11512128B2 (en) | 2011-09-23 | 2022-11-29 | Mereo Biopharma 5, Inc. | VEGF/DLL4 binding agents and uses thereof |
US11339213B2 (en) | 2015-09-23 | 2022-05-24 | Mereo Biopharma 5, Inc. | Methods and compositions for treatment of cancer |
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WO2005054426A3 (en) | 2005-08-04 |
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