# Patent

## Title
Methods of regulating angiogenesis by administering agents which increase apoB-100 polypeptide

## Abstract

A method of treating an angiogenesis related disease or disorder in a subject in need thereof is disclosed. The method comprises administering to the subject a therapeutically effective amount of an agent which regulates an amount of apolipoprotein B (ApoB), and/or an ability of ApoB to transcriptionally control vascular endothelial growth factor receptor 1 (VEGFR1).

## Background
RELATED APPLICATIONS
This application is a National Phase of PCT Patent Application No. PCT/IL2012/050480 having International filing date of Nov. 27, 2012, which claims the benefit of priority under 35 USC §119(e) of U.S. Provisional Patent Application Nos. 61/643,429 filed on May 7, 2012 and 61/563,783 filed on Nov. 27, 2011. The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety.
SEQUENCE LISTING STATEMENT
The ASCII file, entitled 59359SequenceListing.txt, created on May 27, 2014 comprising 47,565 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.
FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to methods of regulating angiogenesis by up or down regulation of Apolipoprotein B (apoB).
Angiogenesis is the formation of new blood vessels from pre-existing vasculature. Angiogenesis is relevant not only to cancer but also to non-neoplastic diseases such as: macular degeneration, psoriasis, endometriosis, arthritis and cardiovascular disease. The growth and metastasis of tumors are dependent upon angiogenesis. Therefore, inhibiting angiogenesis can be used as a method of retarding tumor progression.
Endothelial cells are the building blocks of blood and lymphatic vessels. The interaction between vascular endothelial growth factor (VEGF), which is secreted by tumor cells, and their receptors initiates signaling pathways leading to angiogenesis, including tumor-related angiogenesis. VEGF promotes endothelial cell survival, proliferation, and migration, mainly through the activation of the Flk-1 receptor.
Previous studies have shown that vascular endothelial growth factor receptor 1 (VEGFR1) plays an inhibitory role in angiogenesis, acting as a “sink” for the VEGF ligand [Hiratsuka, S., et al., Proceedings of the National Academy of Sciences of the United States of America 95, 9349-9354 (1998); Kearney, J. B., et al. Blood 99, 2397-2407 (2002); Chappell, J. C., et al., Developmental cell 17, 377-386 (2009)].
Apolipoprotein B (apoB) is a non-exchangeable apolipoprotein found associated exclusively with plasma lipoproteins. In the human genome there is one apob gene of less than 45 kb. In the liver, it is transcribed into a single mRNA of 15 kb and is translated into a single polypeptide of 4536 amino acids called apoB-100. In the intestine, the apoB mRNA is post-transcriptionally edited, resulting in the conversion of a glutamine codon into a stop codon. The edited mRNA is translated into a single polypeptide of 2,152 amino acids called apoB48.
Microsomal triglyceride transfer protein (MTP), an intraluminal protein found within the endoplasmic reticulum of liver and intestine, is required for assembly and secretion of proatherogenic-, apoB-containing lipoproteins such as chylomicrons, very low-density lipoproteins (VLDLs), and low-density lipoproteins (LDLs). Following their assembly as mature particles, apoB-lipoproteins are secreted to the blood and lymph stream by MTP.
U.S. patent applications Ser. No. 20110189300 and 20110003713 teach siRNA molecules that silence ApoB expression and methods of using such siRNAs for the treatment of atherosclerosis, angina pectoris, high blood pressure, diabetes, and hypothyroidism.
U.S. patent application Ser. No. 20050222029 teaches a method of treating cancer by preventing the interaction of ApoB with APOA1.
U.S. patent application Ser. No. 20120053128 teaches a method of inhibiting angiogenesis in mammals using a dimer peptide of apolipoprotein E.
Additional background art includes Salomonsson L. et al., Atherosclerosis. 2003 August; 169(2):259-67.
SUMMARY OF THE INVENTION
According to an aspect of some embodiments of the present invention there is provided a method of treating an angiogenesis related disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent which regulates an amount of apolipoprotein B (ApoB), and/or an ability of ApoB to transcriptionally control vascular endothelial growth factor receptor 1 (VEGFR1), thereby treating the angiogenesis related disease.
According to an aspect of some embodiments of the present invention there is provided a method of identifying a metastasized cancer in a subject in need thereof, comprising determining the level of ApoB in a fluid sample of the subject, wherein an up-regulation of the level of the ApoB compared to the level of ApoB in the fluid sample of a healthy subject, is indicative of a metastasized cancer.
According to an aspect of some embodiments of the present invention there is provided a method of identifying an agent which regulates angiogenesis comprising:
(a) introducing the agent into a cell;
(b) analyzing VEGFR1 transcription in the cell; and
(c) identifying the agent capable of regulating ApoB-dependent VEGFR1 transcription in the cell, thereby selecting the agent which regulates angiogenesis.
According to an aspect of some embodiments of the present invention there is provided a method of identifying an agent which regulates angiogenesis comprising contacting the agent with a polynucleotide which encodes the 3′ untranslated region (UTR) of the ApoB in a cell, wherein the UTR is operatively attached to a reporter molecule, wherein a change in the amount of the reporter molecule is indicative of the agent which regulates angiogenesis.
According to an aspect of some embodiments of the present invention there is provided a method of regulating angiogenesis, the method comprising contacting cells with an agent selected according to the method of the present invention, thereby regulating angiogenesis.
According to some embodiments of the invention, when the regulates is up-regulates, the agent comprises an ApoB polypeptide agent.
According to some embodiments of the invention, when the regulates is up-regulates, the angiogenesis related disease is selected from the group consisting of cancer, arthritis, rheumatoid arthritis, atherosclerotic plaques, corneal graft neovascularization, hypertrophic or keloid scars, proliferative retinopathy, diabetic retinopathy, macular degeneration, granulation, neovascular glaucoma and uveitis.
According to some embodiments of the invention, when the regulates is up-regulates, the angiogenesis related disease is cancer or metastatic cancer.
According to some embodiments of the invention, when the regulates is down-regulates, the agent comprises an antibody directed against the ApoB.
According to some embodiments of the invention, when the regulates is down-regulates, the agent comprises a polynucleotide agent directed against the ApoB.
According to some embodiments of the invention, the polynucleotide agent is selected from the group consisting of an antisense, an siRNA, a DNAzyme and a ribozyme.
According to some embodiments of the invention, when the regulates is down-regulates, the angiogenesis related disease is wound healing, ischemic stroke, ischemic heart disease and gastrointestinal lesions.
According to some embodiments of the invention, the administering is effected in vivo.
According to some embodiments of the invention, the administering is effected ex vivo.
According to some embodiments of the invention, the agent is formulated for ex vivo administration.
According to some embodiments of the invention, the fluid sample is selected from the group consisting of blood, plasma, saliva and urine.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings and images. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
FIGS. 1A-Q illustrate that stl, a zebrafish mutant exhibits excessive angiogenesis. A, Confocal angiography of a 3.5 day post fertilization (dpf) zebrafish, with the subintestinal vessels (SIVs) area shown in (B-D) boxed in red and the intersegmental vessel (ISV) area shown in F,G boxed in yellow. B-D, Confocal images of SIVs in 3.5 dpf (B,C) and 5 dpf (D) wild type (B) and stl mutant embryos (C,D). Note the ectopic angiogenic segments (arrows) that extend ventrally from the normal subintestinal vein (arrowheads). E, Quantitation of the average number (left panel)-, and average total length (middle panel) of ectopic SIV segments, and average width of the normal SIV plexus (right panel) in 3.5 dpf wild type and stl mutant zebrafish. F-G, Confocal images of intersegmental vessels (ISVs) in the mid-trunk of 5 dpf Tg(fli-EGFP)y1 wild type (F) and stl mutant (G) embryos, showing ectopic branching in stl mutants (arrows). H, Quantitation of the average number of ectopic ISV branch points in 5 dpf wild type and stl mutant zebrafish. I, Confocal image of SIVs of a 3.5 dpf stl; Tg(fli-nEGFP)y7 zebrafish, with individual endothelial cell nuclei clearly visible in both the normal plexus (arrowheads) and ectopic sprouts (arrows). J, Quantitation of the average number of endothelial cell nuclei present in either the normal SW plexus or ectopic SIV sprouts of 3.5 dpf Tg(fli-nEGFP)y7 wild type (n=6) or stl mutant larvae (n=7). P=7.3 e−6 (t test) K, Confocal image of SIVs in a 3.5 dpf wild type Tg(fli-EGFP)y1 zebrafish injected with 4 ng of mtp MO, showing formation of ectopic SW sprouts (arrows) similar to those observed in stl mutants. L, Confocal image of SIVs in a 3.5 dpf stl; Tg(fli-EGFP)y1 embryo with 232 pg of mtp mRNA injected into the yolk syncytial layer at dome (approx. 2000-cell) stage, showing reduction in both number and length of ectopic SIV sprouts (arrows) projecting ventrally from the subintestinal vein (arrowheads). M,N, Alkaline phosphatase (AP) staining of SIVs of 3.5 dpf wild type larva that were either treated with DMSO (M) or with DMSO+10 μM atorvastatin (N) at 2.5 dpf. Atorvastatin treatment results in ectopic SW branching. O, Quantitation of the average number (left panel) (nWT+DMSO=41, nWT+Atorvastatin=38) and average total length (right) (nWT+DMSO=39, nWT+Atorvastatin=35), of ectopic SIV segments in DMSO or 10 μM atorvastatin treated larvae *p=1.4 e−4, ** p=7.7 e−5 t-test. P-Q, transmitted light images of 5 dpf Oil Red O (ORO)-stained wild type (P) and stl mutant (Q) larvae, showing strongly decreased lipoprotein levels in the vasculature (arrowheads) of stl mutants. Note normal accumulation of lipids in the yolk of both WT and mutant embryos (arrows). Scale bar in B, C, D, I, K, L 30 μm; in F, G, P, Q 60 μm. All values are mean; error bars show standard deviation of the mean (sdm).
FIGS. 2A-N illustrate that the excess angiogenesis phenotype is not caused by global lipid starvation. A, Transmitted light images of 4 dpf Oil Red O (ORO)-stained apoCII MO-injected larvae, showing increased circulating lipoprotein levels in the dorsal aorta (arrows) and cardinal vein (arrowheads). B-F apoCII morphants show impaired angiogenesis with no ectopic sprouts in the SIVs. B-C, Confocal imaging of SIV in 3.5 dpf Tg(fli-EGFP)y control (B), apoCII (C) MO-injected animals present no ectopic SIV sprouts. D-E, Confocal images of 3.5 dpf control (D) or apoCII MO-injected (E) Tg(fli-EGFP)y zebrafish, showing abnormal trunk ISVs in apoCII morphants. F, Quantitation of trunk ISV's phenotype in control (n=17) and apoCII MO-injected embryos (n=15). Trunk ISV's were classified as 0=no ISV sprouts, 0.5=ISVs up to myoseptum and 1=ISVs up to the Dorsal Longitudinal Anastomotic Vessel (DLAV)). G-K, Free fatty acid supply does not inhibit stl's ectopic sprouts. Alkaline phosphatase (AP) staining of SIVs of 3.5 dpf st/ mutants that were either not treated (G) or treated with 6μg/ml short chain fatty acids (scFA) (C6) (H) intermediate chain fatty acid (icFA) (C12) (I) or long chain fatty acid (lcFA) (J). Treatment with free fatty acids did not inhibit the ectopic vessels phenotype observed in stl mutants. K, Quantitation of the average number (upper panel) and average total length (lower panel) of ectopic SIV segments in 3.5 dpf st/ mutants that were either not treated (n=27) or treated with 6 μg/ml short chain fatty acid (scFA) (C6) (n=29), intermediate chain fatty acid (icFA) (C12) (n=21) or long chain fatty acid (1cFA) (C18) (n=17). (*p =0.517, **p=0.5058, ***p =0.1479) (ANOVA). 1-n, Free fatty acid supply does not rescue the abnormal ISVs phenotype of apoCII morphants. L, Quantitation of trunk ISV's phenotype in apoCII MO-injected embryos that were soaked either in normal medium (n=15) or in medium supplemented with 6 μg/ml short chain fatty acid (scFA) (C6) (n=17). Trunk ISV's were classified as 0=no ISV sprouts, 0.5=ISVs up to myoseptum and 1=ISVs up to DLAV) M, N, Confocal images of trunk ISV's of 3.5 dpf control (M) or apoCII MO -injected (N) embryos treated with 6μg/ml (C6). No phenotypic changes were detected upon scFA treatment (compare e,m). p=0.839 x2 test. Scale bar in A, B, C, G, H, I, J 30 μm; in D, E 60 μm. All values are mean; error bars show standard deviation of the mean (sdm).
FIGS. 3A-P illustrate that lipoprotein levels regulate VEGFR1 expression. A, qRT-PCR measurement of the expression of selected genes in 24 hpf mtp MO injected vs. uninjected zebrafish embryos. Relative expression levels in MO injected embryos are normalized to wild type expression levels. vegfr1 expression is strongly reduced. B, Vegfr1 and Tubulin (control) protein levels in extracts from 3 dpf zebrafish, measured by probing western blots with anti-zebrafish Vegfr1 and anti-Tubulin antibodies. Levels of Vegfr1 protein are reduced in stl and cloche (clo) mutants (clo mutants lack most endothelium) and increased in apoCII morpholino-injected zebrafish compared to wild type siblings. C, Levels of VEGFR1 and VEGFR2 protein in confluent HUVECs, cultured in LDL-depleted media with or without addition of LDL (5 or 10 μg/ml). Actin protein was probed as a control. Only VEGFR1 expression is upregulated in HUVECs treated with LDL. D-H, Endothelial VEGFR1 expression is upregulated in hyperlipidemic mice. D-G, Confocal images of the aortic root of wild type C57B1/6 sibling (D) or ApoE null (E) mice, and abdominal Aorta of wild type C57B1/6 sibling (F) or LDLR null (G) mice, stained with Pecam (D-G) and anti-VEGFR1 antibody (D′-G′). VEGFR1 is specifically upregulated in endothelial cells of hyperlipidemic mice as denoted by co-localization with Pecam staining (D′′-G′′). Scale bar presented in G′′ refers to images D-G, 25 μm. H, Semi-quantitative RT-PCR measurement of vegfr1 expression in aortic arches dissected from wild type sibling (n=4) or apoE null (n=4) mice. p=4 e−14 z test. I-K, vegfr1 inhibits excessive angiogenesis in stl mutants. I, J, Transmitted light images of AP stained SIVs in 3.5 dpf stl mutants (I) or stl mutants injected with vegfr1 mRNA (J), showing that vegfr1 overexpression suppresses ectopic sprouting in stl mutants. K, Quantitation of the average number (left panel)-, and average total length (middle panel) of ectopic SIV segments, and average width of the normal SIV plexus (right panel) in 3.5 dpf stl mutants (n=26) and stl mutants injected with vegfr1 mRNA (n=23). *p=1.6 e−4, **p=2.6 e−4, ***p=0.21 t test. L, M, LDL exposure suppresses VEGFR-mediated endothelial migration. HUVECs exposed to either siRNA control (non-targeting) or siRNA for VEGFR1 were plated to confluency for wound assays. A 200 um wound was inflicted in both groups and each was treated with either LDL or vehicle. Migration of cells was recorded for 24 hrs (note proliferation cycle of these cells ranges from 36 to 48 hrs). Four wells per experimental group were measured. Results were normalized to control and expressed as migration area over control. Bars represent mean (+/− SE). N-P, vegfr1 is transcriptionally regulated. N, O, Confocal images of Tg(flt1:YFP)hu4624 wt (N) and mtp MO injected (O) zebrafish. YFP expression is downregulated in mtp morphants. P, qRT-PCR measurements of the relative expression levels of YFP in 48 hpf wt and mtp MO injected Tg(flt1:YFP)hu4624/+ zebrafish. The levels of yfp mRNA are strongly reduced in mtp morphants. p=0.0248 t test. Scale bar in I, J, 30 μm, in N, O, 60 μm.
FIGS. 4A-L illustrate that apoB particles regulate angiogenesis by directly acting on endothelial cells. A, B, DiI-LDL inhibits excessive angiogenesis in stl mutants. Alkaline phosphatase (AP) staining of SIVs of 3.5 dpf stl mutants that were either not injected (n=27) (A) or injected intravascularly with DiI-LDL (n=35) (B) at 2.5 dpf. Note the lack of ectopic sprouts observed in treated embryos. C, Quantitation of the average number (left panel) and average total length (right segment) of ectopic SIV segments in 3.5 dpf stl mutants that were either not injected or injected with DiI-LDL. *p=0.0001 **p=3 e−6 t test. D, Diagram illustrating the procedure used for transplanting transfected HEK293 cells into the extracellular space between the yolk cell membrane and the outer periderm of zebrafish larva, in close proximity to the SIVs. E,F, Confocal images of the SIVs (green) of 3.5 dpf Tg(fli EGFP)y1 zebrafish transplanted with either MTP and ApoB34 transfected (E)-, or untransfected control (F)-HEK293 cells (red), on one side of the yolk ball. Both, control-untransplanted (E, F right), and transplanted (E, F left) sides of the animal are shown. Only lipoprotein-secreting cells impeded the migration of endothelial cells and the growth of ectopic sprouts in the area of transplantation (E, left panel). G, Quantitation of the overlapping area between SIV endothelium and HEK293 cells in 3.5 dpf transplanted zebrafish as a function of the total length of the interface between the two cell populations. H-K, apoB but not short chain fatty acid (scFA) inhibits excessive angiogenesis in stl mutants. AP staining of SIVs of 4.5 dpf stl mutants that were either not injected (n=16) (H) or injected intravascularly with 6 μg/ml short chain fatty acid (scFA) (n=10) (I) or a delipidated form of apoB-100 (n=24) (J) at 2.5 dpf. Note the lack of ectopic sprouts observed in apoB-injected embryos. K, Quantitation of the average number (left panel) and average total length (right segment) of ectopic SIV segments in 3.5 dpf stl mutants that were either not injected or injected intravascularly with 6 μg/ml short chain fatty acid or a delipidated form of apoB-100. *p=0.0411 **p=4.6 e−5 t test 1, Schematic model illustrating the effects of circulating lipoproteins on angiogenesis. Scale bar in A, B, H, I, J 30 μm. Scale bar presented in F (right panel) refers to images E, F, 60 μm. n.s, not significant.
FIGS. 5A-F illustrate the development of the subintestinal vessels (SIVs) and absorption of yolk. A, B, Zebrafish SIVs begin to form at 2.5 dpf, from vascular sprouts that emerge from the supraintestinal artery (SIA) at the ventral midline of the trunk and then grow bilaterally onto the dorsolateral surface of the large yolk cell of the fish (AMAAnterior Mesenteric Artery, PVC—Posterior Cardinal Vein, DA—Dorsal Aorta). C. At 4 dpf the subintestinal vein (SIV) delimits the ventral edge of this vascular bed. The most rostral unbranched portion of the left SIV will become the hepatic portal vein (HPV). D. The yolk is gradually absorbed to supply the growing larva, and by 7 dpf the SW s surround and perfuse the well-differentiated intestine, liver (L) swim bladder (SB) and pancreatic anlagen. E, F, Yolk is absorbed in wild type animals (E), but remains rounded and unabsorbed in stalactite (stl) mutants (F). Scale bar 60 μm.
FIG. 6 illustrates quantitation of SW vascular plexus width and ectopic ventral SW sprout number and length. The maximal width of the normal SIV plexus is measured from supraintestinal artery to subintestinal vein (1). The number of ectopic sprouts greater than 1 μm in length projecting ventrally from the subintestinal vein is counted, and the total length of all of these sprouts (2) is measured. Measurements are performed on the SIV vessels of at least 10 animals per treatment, and standard errors of the mean are calculated for these measurements and used in the plots displayed. Scale bar 30 μm.
FIGS. 7A-C illustrates the defect in the microsomal triglyceride transfer protein (mtp) gene, identified by molecular cloning of the stl mutation. A. Interval in linkage group 1 containing the mtp gene. Polymorphic markers used in this study are shown above the map, whereas numbers below the map indicate number of recombinants over the number of informative meioses. B. Sequence analysis of mtp cDNA from stl mutants and wild type siblings reveals a T=>C base change causing a Leu475=>Pro change in the encoded polypeptide. c. Leucine 475 is a highly conserved amino acid residue in the MTP polypeptide.
FIGS. 8A-G illustrates the phenotype of stl mutants. A, Quantitation of the percentage of 3.5 dpf Tg(fli-EGFP)y uninjected control (n=97) or mtp MO injected (n=98) zebrafish displaying mutant phenotype (ectopic SW sprouts). b, Quantitation of the percentage of larvae from an stl/+ incross (25% of embryos are stl/stl) that display mutant phenotype (ectopic SIV sprouts) with either no mRNA injected (n=104), or 232 pg mtp mRNA injected into the 1-cell stage cytoplasm (n=49) or injected into the dome stage yolk syncytial layer (n=29). C, Confocal angiography of a 3.5 dpf zebrafish, with the subintestinal vessels (SIVs) area shown in (D-F) below boxed in red. (D-F) Confocal images of the SIVs in 3.5 dpf Tg(fli-EGFP)y1 zebrafish un-uninjected (D) or injected with either apoB (E) or apoA1 Morpholinos (F). Ectopic SIV sprouts are observed in apoB MO-injected embryos, reminiscent of those seen in stl mutants. apoA1 morphants, however, do not exhibit any morphological change even when injected with high concentration of morpholino. G, mtp is not expressed by zebrafish EC's. GFP+ ECs and GFP− cells from 3.5 dpf Tg(fli-EGFP)y1 zebrafish were FACS sorted and their mRNA was used for semi-quantitative RT-PCR. mtp was found to be expressed by GFP− cells but not by GFP+ ECs. Scale bar in C—60 μm; in D, E, F 30 μm.
FIGS. 9A-C are graphs illustrating that the cells of stl mutants and apoCII morphants are starving for lipids. (A, B) Dechorionaized 24 hpf stl mutants, apoCII MO-injected and WT embryos were soaked in 2 μCi/ml3 Holeic acid for 48 hours. Embryo lipid extracts were developed on silica gel 60 TLC plates and spots corresponding to triglyceride standards were quantified. Exogenously supplied radiolabeled fatty acids were incorporated at higher rates in both stl mutants (n=93) and ApoCII morphants (n=60) as opposed to WT embryos (n=93 in A and n=65 in B), indicating that cells are lipid-starved in both models. Pa=2 e−12, Pb=8 e−17; t test. (C) Total cholesterol levels in wild type, apo CII morphants and stl mutants were analyzed using gas chromatography-mass spectrometry (GC-MS). Cholesterol levels are reduced in apoCII morphants and stl mutants when compared to WT siblings.
FIGS. 10A-D are photographs illustrating that downregulation of lpl results in poorly developed vasculature with no ectopic SIV sprouts. A,B, Confocal imaging of SIV in 3.5 dpf Tg(fli-EGFP)y1 control (A) or lpl MO-injected (B) animals present no ectopic SIV sprouts. C, D, Trunk ISVs in 3.5 dpf Tg(fli-EGFP)y control (C) or lpl-MO injected zebrafish larvae. Lpl morphants display short and partially lumenized ISVs.
FIG. 11 is a survival curve of WT zebrafish embryos treated with increasing concentrations of oleic acid. Only embryos treated with less than 6 μg/ml oleic acid survived until 6 dpf.
FIG. 12 illustrates vegfr1 expression is specifically reduced in mtp-MO injected zebrafish. In situ hybridization of control un-injected (left panels) or mtp MO-injected (right panels) 24 hpf zebrafish, probed for vegfr1, vegfr2, vegfr3, or veCad showing specific increase in vegfr1 mRNA levels. Scale bar 60 μm.
FIGS. 13A-E illustrate the vessels of control or vegfr1 morpholino-injected animals. A, C, Confocal imaging of SIVs in 3.5 dpf Tg(fli-EGFP)y control (A) or vegfr1 (C) morpholino-injected animals, showing ectopic SIV sprouts in vegfr1 morphants (arrows) similar to these observed in stl mutants. B, D. Confocal images of vessels in the mid-trunk of the same embryos showing ectopic branching and sprouting (arrowheads) in vegfr1 MO-injected embryo. E. Quantitation of the average number (left panel), average total length (middle panel) of ectopic SW segments, and average width of the normal SIV plexus (right panel) in 3.5 dpf control and vegfr1 MO-injected zebrafish larvae. Scale bar in A, C 30 μm; in B, D 60 μm.
FIGS. 14A-B illustrates that Huh7MTP promotes the lipidation and the secretion of apoB34. A, ApoB34 was transfected into HEK293 cells without (−) or with (+) Huh7MTP, as indicated. Twenty-four hours posttransfection cells were radiolabeled with 35[S]Met and Cys for 3 hours and cell lysate (C) and media (M) samples subjected to immunoprecipitation with anti-apoB antibody, followed by 8% SDS-PAGE and phosphorimager analysis. Cotransfection with Huh7MTP resulted in an  ̃5-fold increase in apoB34 secretion relative to control cells (compare lanes 2 and 4). Huh7MTP interacted with apoB intracellularly as evidenced by its co-immunoprecipitation with anti-apoB antibody (lane 3). B. Huh7MTP promotes the lipidation of ApoB22. ApoB22 was cotransfected without (−) or with (+) Huh7MTP, as indicated. 24 hours post-transfection, cells were radiolabeled with 35[S]Met and Cys for 3 hours. Media was adjusted to 1.25 g/ml with KBr and subjected to equilibrium density gradient centrifugation. The top (T) buoyant fractions and bottom (B) lipid-poor infranatant fractions were subjected to immunoprecipitation with anti-apoB anybody, fractionation by 12.5% SDS-PAGE and phosphorimager analysis. ApoB22 was found in the top buoyant fraction only upon coexpression with Huh7MTP (compare lanes 1 and 3).
FIG. 15 is a quantitative analysis of HEK293 cell transplants. Screen output illustrating the use of a software tool developed for the purpose of quantifying the effects of exogenous secreted lipoproteins on zebrafish blood vessels. Yolk region of a Tg(fli:EGFP)y1 embryo, showing transplanted HEK293 cells (red) and subintestinal vessels (green). Phenotypic quantitation was performed at 3.5 dpf only in embryos that had been successfully transplanted with fluorescent red cells adjacent to green fluorescent endothelial cells. The total length of the interface between endothelial and transplanted cells (L1, blue), as well as the total length of overlapping between the two cell populations (L2, yellow) were traced and measured. We then used the ratio r=L2/(L1+L2) as a measure of the tendency of endothelial cells to penetrate the ApoB secreting cells area (once in their vicinity). The ratios were computed for three transplanted animals per treatment (transfected and untransfected HEK293 cells), and were compared using t-test.
FIGS. 16A-B illustrates that LDL suppresses endothelial cell migration. HUVEC or HAEC migration assays were carried out in a Boyden Chamber in either LDL depleted serum (LDL(−)) or LDL(−) serum supplemented with LDL. All assays were performed in triplicate, measuring the number of transwell transmigrated cells, and standard deviations of the data were calculated. Addition of LDL to LDL(−) serum resulted in decreased migration of either HUVEC or HAEC. B. Migration was assessed by endothelial wound assays. Confluent endothelial monolayers were wounded with a 10 μl pipet tip. The wound was washed once with serum free media followed by incubation with media containing either LDL (10 μg/ml); 1% LDL-depleted serum or 1% LDL-depleted serum supplemented with 10 μg/ml of LDL. Cells under indicated treatment were imaged by time-lapse video microscopy. To calculate the cell migration rate, the wound area was determined prior and after completion of the experiment (8 hours). The graph represents four independent experiments in triplicates. Bars showed standard deviation. P=0.03. C, HUVECs were incubated with either 20% LPDS or 20% LPDS supplemented with LDL 10 μg/ml or 25 Vg/ml for indicated periods. The number of cells was then determined using the MTT cell growth assay. HUVEC proliferation is not affected by addition of LDL to LPDS medium.



DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to methods of regulating angiogenesis by up or down regulation of Apolipoprotein B (apoB).
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
Despite the clear major contribution of hyperlipidemia to the prevalence of cardiovascular disease in the developed world, the direct effects of lipoproteins on endothelial cells have so far remained obscure and controversial.
The present inventors have now discovered a novel mechanism of vessel growth modulation by lipoprotein availability. Using novel genetic mutants, morpholino knockdown, and xenografts to manipulate lipoprotein levels in zebrafish, in combination with hyperlipidemic mice and cultured endothelial cells, the present inventors have demonstrated that apoB-lipoproteins negatively regulate angiogenesis (FIGS. 1A-Q).
Further the present inventors have provided mechanistic data highlighting VEGFR1, which acts as a decoy receptor for VEGF, as a key mediator of the endothelial response to lipoproteins (FIGS. 3A-P).
Finally the present inventors have shown that the apoB protein particle, but not the lipid moieties within apoB-lipoproteins, plays a major role in triggering the vascular response (FIGS. 4H-K).
Taken together, these findings define an important new pathway controlling the angiogenic switch, and open new avenues for the treatment of lipoprotein-related vascular disorders.
Thus, according to one aspect of the present invention, there is provided a method of treating an angiogenesis related disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent which regulates an amount of apolipoprotein B (ApoB), and/or an ability of ApoB to transcriptionally regulate vascular endothelial growth factor receptor 1 (VEGFR1), thereby treating the angiogenesis related disease.
As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
As used herein the term “subject” refers to any (e.g., mammalian) subject, preferably a human subject.
As used herein, the term “angiogenesis” refers to the generation of new blood vessels into a tissue or organ. Under normal physiological conditions, humans or animals only undergo angiogenesis in very specific restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonal development and formation of the corpus luteum, endometrium and placenta. The control of angiogenesis is a highly regulated system of angiogenic stimulators and inhibitors. The control of angiogenesis has been found to be altered in certain disease states and, in many cases, the pathological damage associated with the disease is related to the uncontrolled angiogenesis.
Angiogenesis-related diseases include, but are not limited to, inflammatory, autoimmune, and infectious diseases; angiogenesis-dependent cancer, including, for example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis; eczema; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; wound granulation; intestinal adhesions, atherosclerosis, scleroderma, wound healing, ischemic stroke, ischemic heart disease, gastrointestinal lesions, warts, and hypertrophic scars (i.e., keloids). The disease may have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa), ulcers (Helobacter pylori), tuberculosis, and leprosy.
Other examples of cancer for which the ApoB related agent may be administered according to this aspect of the present invention include, but are not limited to, adrenocortical carcinoma, hereditary; bladder cancer; breast cancer; breast cancer, ductal; breast cancer, invasive intraductal; breast cancer, sporadic; breast cancer, susceptibility to; breast cancer, type 4; breast cancer, type 4; breast cancer-1; breast cancer-3; breast-ovarian cancer; Burkitt's lymphoma; cervical carcinoma; colorectal adenoma; colorectal cancer; colorectal cancer, hereditary nonpolyposis, type 1; colorectal cancer, hereditary nonpolyposis, type 2; colorectal cancer, hereditary nonpolyposis, type 3; colorectal cancer, hereditary nonpolyposis, type 6; colorectal cancer, hereditary nonpolyposis, type 7; dermatofibrosarcoma protuberans; endometrial carcinoma; esophageal cancer; gastric cancer, fibrosarcoma, glioblastoma multiforme; glomus tumors, multiple; hepatoblastoma; hepatocellular cancer; hepatocellular carcinoma; leukemia, acute lymphoblastic; leukemia, acute myeloid; leukemia, acute myeloid, with eosinophilia; leukemia, acute nonlymphocytic; leukemia, chronic myeloid; Li-Fraumeni syndrome; liposarcoma, lung cancer; lung cancer, small cell; lymphoma, non-Hodgkin's; lynch cancer family syndrome II; male germ cell tumor; mast cell leukemia; medullary thyroid; medulloblastoma; melanoma, meningioma; multiple endocrine neoplasia; myeloid malignancy, predisposition to; myxosarcoma, neuroblastoma; osteosarcoma; ovarian cancer; ovarian cancer, serous; ovarian carcinoma; ovarian sex cord tumors; pancreatic cancer; pancreatic endocrine tumors; paraganglioma, familial nonchromaffin; pilomatricoma; pituitary tumor, invasive; prostate adenocarcinoma; prostate cancer; renal cell carcinoma, papillary, familial and sporadic; retinoblastoma; rhabdoid predisposition syndrome, familial; rhabdoid tumors; rhabdomyosarcoma; small-cell cancer of lung; soft tissue sarcoma, squamous cell carcinoma, head and neck; T-cell acute lymphoblastic leukemia; Turcot syndrome with glioblastoma; tylosis with esophageal cancer; uterine cervix carcinoma, Wilms' tumor, type 2; and Wilms' tumor, type 1, and the like.
As used herein, the phrase “down-regulating angiogenesis” refers to either slowing down or stopping the angiogenic process, which leads to formation of new blood vessels. The phrase “upregulating angiogenesis” refers to enhancing the expression of a dormant or minimally-functioning endothelial cell angiogenesis activator.
The term “Apolipoprotein B” or “ApoB”, Swiss-Prot number P04114, refers to at least an active portion of ApoB (i.e., a portion having ApoB activity), which is the main apolipoprotein of chylomicrons and low density lipoproteins (LDL).
As used herein the phrase “ApoB activity” refers to at least the transcriptional activity of ApoB i.e., the ability of ApoB to up-regulate transcription of the target gene—-VEGFR1. The transcriptional activity may be a direct activity on the VEGFR1 gene or a non-direct activity via an intermediate molecule/molecules. According to one embodiment, the ApoB comprises an amino acid sequence of a functional DNA binding domain.
ApoB may be found in the plasma in 2 main forms: apoB48 and apoB 100, which are synthesized in the intestine and liver, respectively, due to an organ-specific stop codon. ApoB48 contains 2,152 residues compared to 4,535 residues in apoB100. Cloning and characterization of APOB is described by, e.g., Glickman et al., PNAS USA 83:5296-5300 (1986); Chen et al., J. Biol. Chem. 261: 2918-12921 (1986); and Hospattankar et al., J. Biol. Chem. 261:9102-9104 (1986).
APOB sequences are set forth in, e.g., GenBank Accession Nos. NM_000384 and additional sequences such as AB208846.1, AJ399514.1 AK290844.1, BC051278.1, HM487065.1, J02610.1, K03175.1, M10374.1, M12413.1, M12480.1, M12681.1, M14081.1, M14162.1, M15421.1, M17367.1, M17779.1, M18036.1, M18471.1, M19734.1, M31030.1, M36676.1, X03045.1, X03324.1, X03325.1, X03326.1, X04506.1 and X04714.1.
The term “Vascular endothelial growth factor receptor 1” or “VEGFR1” refers to the receptor tyrosine kinases (RTKs) which contains an extracellular ligand-binding region with seven immunoglobulin (Ig)-like domains, a transmembrane segment, and a tyrosine kinase (TK) domain within the cytoplasmic domain. This protein binds to VEGFR-A, VEGFR-B and placental growth factor and plays an important role in angiogenesis and vasculogenesis. VEGFR1 may be encoded by any of the following four alternative transcripts: NM_001159920.1, NM_001160030.1, NM_001160031.1, or NM_002019.4.
Agents which increase the amount of ApoB include agents which are capable of increasing the transcription (for example a transcription factor known to interact with the 5′ untranslated region of ApoB) of ApoB, the translation of ApoB or the stability of ApoB. Additionally, the agent which increases the amount of ApoB, may be a polynucleotide which encodes ApoB, the protein itself or an active peptide thereof.
As mentioned, the present invention also contemplates agents which are capable of increasing the ability of ApoB to transcriptionally upregulate VEGFR1 for decreasing angiogenesis. Such agents may act downstream of ApoB in order to induce transcription of VEGFR1.
All of the above described agents are useful for treating diseases where a decrease of angiogenesis is required. Such diseases include, but are not limited to cancer, arthritis, rheumatoid arthritis, atherosclerotic plaques, corneal graft neovascularization, hypertrophic or keloid scars, proliferative retinopathy, diabetic retinopathy, macular degeneration, granulation, neovascular glaucoma and uveitis.
Preferably the ApoB polypeptide (or the polynucleotide which encodes the ApoB polypeptide) which is administered to the subject is (or encodes a protein that is) at least 50% homologous, more preferably at least 60% homologous, more preferably at least 70% homologous, more preferably at least 80% homologous, and most preferably at least 90% homologous to the polypeptide sequence as set forth in SEQ ID NO:34 as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters) comprising ApoB activity. The homolog may also refer to a deletion, insertion, or substitution variant, including an amino acid substitution, thereof and biologically active polypeptide fragments thereof.
Recombinant techniques are typically used to generate the ApoB polypeptides of the present invention. These techniques may be preferred due to the number of amino acids in ApoB polypeptides and the large amounts required thereof. Such recombinant techniques are described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.
To produce an expression vector for the expression of the ApoB of the present invention, a polynucleotide encoding the ApoB of the present invention is ligated into a nucleic acid expression vector, which comprises the polynucleotide sequence under the transcriptional control of a cis-regulatory sequence (e.g., promoter sequence) suitable for directing constitutive, tissue specific or inducible transcription of the ApoB of the present invention in the host cells.
The phrase “an isolated polynucleotide” refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
As used herein the phrase “complementary polynucleotide sequence” refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase.
As used herein the phrase “genomic polynucleotide sequence” refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
As used herein the phrase “composite polynucleotide sequence” refers to a sequence, which is at least partially complementary and at least partially genomic. A composite sequence can include some exonal sequences required to encode the ApoB of the present invention, as well as some intronic sequences interposing there between. The intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.
As mentioned hereinabove, polynucleotide sequences of the present invention are inserted into expression vectors (i.e., a nucleic acid construct) to enable expression of the recombinant ApoB. The expression vector of the present invention may include additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). Typical cloning vectors contain transcription and translation initiation sequences (e.g., promoters, enhances) and transcription and translation terminators (e.g., polyadenylation signals).
A variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the ApoBs of the present invention. These include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the ApoB coding sequence; yeast transformed with recombinant yeast expression vectors containing the ApoB coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the ApoB coding sequence.
Other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the ApoB), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed ApoB.
Various methods can be used to introduce the expression vector of the present invention into the host cell system. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.
Transformed cells are cultured under effective conditions, which allow for the expression of high amounts of recombinant peptide. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective medium refers to any medium in which a cell is cultured to produce the recombinant ApoB of the present invention. Such a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
Depending on the vector and host system used for production, resultant ApoB of the present invention may either remain within the recombinant cell, secreted into the fermentation medium, secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or retained on the outer surface of a cell or viral membrane.
Following a predetermined time in culture, recovery of the recombinant ApoB is effected.
The phrase “recovering the recombinant ApoB” used herein refers to collecting the whole fermentation medium containing the ApoB and need not imply additional steps of separation or purification.
Thus, the ApoB polypeptides of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
To facilitate recovery, the expressed coding sequence can be engineered to encode an ApoB fused to a cleavable moiety. Such a fusion protein can be designed so that the ApoB can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the cleavable moiety. Where a cleavage site is engineered between the ApoB and the cleavable moiety, the ApoB can be released from the chromatographic column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].
The ApoB of the present invention is preferably retrieved in “substantially pure” form.
As used herein, the phrase “substantially pure” refers to a purity that allows for the effective use of the ApoB in the applications described herein.
In addition to being synthesizable in host cells, the ApoB of the present invention can also be synthesized using in vitro expression systems. These methods are well known in the art and the components of the system are commercially available.
As mentioned, the ApoB may be administered to the subject in need thereof as polynucleotides where they are expressed in vivo i.e. gene therapy.
The phrase “gene therapy” as used herein refers to the transfer of genetic material (e.g. DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition or phenotype. The genetic material of interest encodes a product (e.g. a protein, polypeptide, peptide, functional RNA, antisense) whose production in vivo is desired. For example, the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value. For review see, in general, the text “Gene Therapy” (Advanced in Pharmacology 40, Academic Press, 1997).
Two basic approaches to gene therapy have evolved: (1) ex vivo and (2) in vivo gene therapy. In ex vivo gene therapy cells are removed from a patient, and while being cultured are treated in vitro. Generally, a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the hostlpatient. These genetically reimplanted cells have been shown to express the transfected genetic material in situ. The cells may be autologous or non-autologous to the subject. Since non-autologous cells are likely to induce an immune reaction when administered to the body several approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include either suppressing the recipient immune system or encapsulating the non-autologous cells in immunoisolating, semipermeable membranes before transplantation.
In in vivo gene therapy, target cells are not removed from the subject rather the genetic material to be transferred is introduced into the cells of the recipient organism in situ, that is within the recipient. These genetically altered cells have been shown to express the transfected genetic material in situ.
To confer specificity, preferably the nucleic acid constructs used to express the ApoB of the present invention comprise cell-specific promoter sequence elements.
Recombinant viral vectors are useful for in vivo expression of the ApoB of the present invention since they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
As mentioned, the present invention also contemplates agents which are capable of decreasing an amount of ApoB for enhancing angiogenesis. Such agents are useful for treating diseases such as in atherosclerosis induced coronary artery blockage (e.g., angina pectoris), in necrotic damage following accidental injury or surgery, or in gastrointestinal lesions such as ulcers.
Following is a list of agents capable of downregulating expression of ApoB.
One example, of an agent capable of downregulating ApoB is an antibody or antibody fragment capable of specifically binding thereto. Preferably, the antibody specifically binds at least one epitope of ApoB. As used herein, the term “epitope” refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
The term “antibody” as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab′)2, and Fv that are capable of binding to macrophages. These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab′, the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule; (3) (Fab′)2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody (“SCA”), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
Downregulation of ApoB can be also achieved by RNA silencing. As used herein, the phrase “RNA silencing” refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or “silencing” of the expression of a corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.
As used herein, the term “RNA silencing agent” refers to an RNA which is capable of specifically inhibiting or “silencing” the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g, the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include noncoding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs. In one embodiment, the RNA silencing agent is capable of inducing RNA interference. In another embodiment, the RNA silencing agent is capable of mediating translational repression.
According to an embodiment of the invention, the RNA silencing agent is specific to the target RNA (e.g., ApoB) and does not cross inhibit or silence a gene or a splice variant which exhibits 99% or less global homology to the target gene, e.g., less than 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% global homology to the target gene.
RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla. Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA.
The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex.
Accordingly, some embodiments of the invention contemplate use of dsRNA to downregulate protein expression from mRNA.
According to one embodiment, the dsRNA is greater than 30 bp. The use of long dsRNAs (i.e. dsRNA greater than 30 bp) has been very limited owing to the belief that these longer regions of double stranded RNA will result in the induction of the interferon and PKR response. However, the use of long dsRNAs can provide numerous advantages in that the cell can select the optimal silencing sequence alleviating the need to test numerous siRNAs; long dsRNAs will allow for silencing libraries to have less complexity than would be necessary for siRNAs; and, perhaps most importantly, long dsRNA could prevent viral escape mutations when used as therapeutics.
Various studies demonstrate that long dsRNAs can be used to silence gene expression without inducing the stress response or causing significant off-target effects—see for example [Strat et al., Nucleic Acids Research, 2006, Vol. 34, No. 13 3803-3810; Bhargava A et al. Brain Res. Protoc. 2004; 13:115-125; Diallo M., et al., Oligonucleotides. 2003; 13:381-392; Paddison P. J., et al., Proc. Natl Acad. Sci. USA. 2002; 99:1443-1448; Tran N., et al., FEBS Lett. 2004; 573:127-134].
In particular, the invention according to some embodiments thereof contemplates introduction of long dsRNA (over 30 base transcripts) for gene silencing in cells where the interferon pathway is not activated (e.g. embryonic cells and oocytes) see for example Billy et al., PNAS 2001, Vol 98, pages 14428-14433. and Diallo et al, Oligonucleotides, Oct. 1, 2003, 13(5): 381-392. doi:10.1089/154545703322617069.
The invention according to some embodiments thereof also contemplates introduction of long dsRNA specifically designed not to induce the interferon and PKR pathways for down-regulating gene expression. For example, Shinagwa and Ishii [Genes & Dev. 17 (11): 1340-1345, 2003] have developed a vector, named pDECAP, to express long double-strand RNA from an RNA polymerase II (Pol II) promoter. Because the transcripts from pDECAP lack both the 5′-cap structure and the 3′-poly(A) tail that facilitate ds-RNA export to the cytoplasm, long ds-RNA from pDECAP does not induce the interferon response.
Another method of evading the interferon and PKR pathways in mammalian systems is by introduction of small inhibitory RNAs (siRNAs) either via transfection or endogenous expression.
The term “siRNA” refers to small inhibitory RNA duplexes (generally between 18-30 basepairs) that induce the RNA interference (RNAi) pathway. Typically, siRNAs are chemically synthesized as 21mers with a central 19 bp duplex region and symmetric 2-base 3′-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a 100-fold increase in potency compared with 21mers at the same location. The observed increased potency obtained using longer RNAs in triggering RNAi is theorized to result from providing Dicer with a substrate (27mer) instead of a product (21mer) and that this improves the rate or efficiency of entry of the siRNA duplex into RISC.
It has been found that position of the 3′-overhang influences potency of an siRNA and asymmetric duplexes having a 3′-overhang on the antisense strand are generally more potent than those with the 3′-overhang on the sense strand (Rose et al., 2005). This can be attributed to asymmetrical strand loading into RISC, as the opposite efficacy patterns are observed when targeting the antisense transcript.
The strands of a double-stranded interfering RNA (e.g., an siRNA) may be connected to form a hairpin or stem-loop structure (e.g., an shRNA). Thus, as mentioned the RNA silencing agent of some embodiments of the invention may also be a short hairpin RNA (shRNA).
The term “shRNA”, as used herein, refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop. Examples of oligonucleotide sequences that can be used to form the loop include 5′-UUCAAGAGA-3′ (Brummelkamp, T. R. et al. (2002) Science 296: 550) and 5′-UUUGUGUAG-3′ (Castanotto, D. et al. (2002) RNA 8:1454). It will be recognized by one of skill in the art that the resulting single chain oligonucleotide forms a stem-loop or hairpin structure comprising a double-stranded region capable of interacting with the RNAi machinery.
According to another embodiment the RNA silencing agent may be a miRNA. miRNAs are small RNAs made from genes encoding primary transcripts of various sizes. They have been identified in both animals and plants. The primary transcript (termed the “pri-miRNA”) is processed through various nucleolytic steps to a shorter precursor miRNA, or “pre-miRNA.” The pre-miRNA is present in a folded form so that the final (mature) miRNA is present in a duplex, the two strands being referred to as the miRNA (the strand that will eventually basepair with the target) The pre-miRNA is a substrate for a form of dicer that removes the miRNA duplex from the precursor, after which, similarly to siRNAs, the duplex can be taken into the RISC complex. It has been demonstrated that miRNAs can be transgenically expressed and be effective through expression of a precursor form, rather than the entire primary form (Parizotto et al. (2004) Genes & Development 18:2237-2242 and Guo et al. (2005) Plant Cell 17:1376-1386).
Unlike, siRNAs, miRNAs bind to transcript sequences with only partial complementarity (Zeng et al., 2002, Molec. Cell 9:1327-1333) and repress translation without affecting steady-state RNA levels (Lee et al., 1993, Cell 75:843-854; Wightman et al., 1993, Cell 75:855-862). Both miRNAs and siRNAs are processed by Dicer and associate with components of the RNA-induced silencing complex (Hutvagner et al., 2001, Science 293:834-838; Grishok et al., 2001, Cell 106: 23-34; Ketting et al., 2001, Genes Dev. 15:2654-2659; Williams et al., 2002, Proc. Natl. Acad. Sci. USA 99:6889-6894; Hammond et al., 2001, Science 293:1146-1150; Mourlatos et al., 2002, Genes Dev. 16:720-728). A recent report (Hutvagner et al., 2002, Sciencexpress 297:2056-2060) hypothesizes that gene regulation through the miRNA pathway versus the siRNA pathway is determined solely by the degree of complementarity to the target transcript. It is speculated that siRNAs with only partial identity to the mRNA target will function in translational repression, similar to an miRNA, rather than triggering RNA degradation.
Synthesis of RNA silencing agents suitable for use with some embodiments of the invention can be effected as follows. First, the selected mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3′ adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245]. It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5′ UTR mediated about 90% decrease in cellular GAPDH mRNA and completely abolished protein level (wwwdotambiondotcom/techlib/tn/91/912dothtml).
Second, potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (wwwdotncbidotnlmdotnihdotgov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out.
Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55%. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
It will be appreciated that the RNA silencing agent of some embodiments of the invention need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides.
In some embodiments, the RNA silencing agent provided herein can be functionally associated with a “cell-penetrating peptide.” As used herein, a “cell-penetrating peptide” is a peptide that comprises a short (about 12-30 residues) amino acid sequence or functional motif that confers the energy-independent (i.e., non-endocytotic) translocation properties associated with transport of the membrane-permeable complex across the plasma and/or nuclear membranes of a cell. The cell-penetrating peptide used in the membrane-permeable complex of some embodiments of the invention preferably comprises at least one non-functional cysteine residue, which is either free or derivatized to form a disulfide link with a double-stranded ribonucleic acid that has been modified for such linkage. Representative amino acid motifs conferring such properties are listed in U.S. Pat. No. 6,348,185, the contents of which are expressly incorporated herein by reference. The cell-penetrating peptides of some embodiments of the invention preferably include, but are not limited to, penetratin, transportan, pIsl, TAT(48-60), pVEC, MTS, and MAP.
Another agent capable of downregulating ApoB is a DNAzyme molecule capable of specifically cleaving an mRNA transcript or DNA sequence of the SLUG. DNAzymes are single-stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R. R. and Joyce, G. Chemistry and Biology 1995; 2:655; Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997; 943:4262) A general model (the “10-23” model) for the DNAzyme has been proposed. “10-23” DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions (Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian, L M [Curr Opin Mol Ther 4:119-21 (2002)].
Examples of construction and amplification of synthetic, engineered DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes of similar design directed against the human Urokinase receptor were recently observed to inhibit Urokinase receptor expression, and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al, 20002, Abstract 409, Ann Meeting Am Soc Gen Ther wwwdotasgtdotorg). In another application, DNAzymes complementary to bcr-ab1 oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of CML and ALL.
Downregulation of ApoB can also be effected by using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding ApoB.
Another agent capable of downregulating ApoB is a ribozyme molecule capable of specifically cleaving an mRNA transcript encoding ApoB. Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)]. The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications.
An additional method of regulating the expression of ApoB in cells is via triplex forming oligonuclotides (TFOs). Recent studies have shown that TFOs can be designed which can recognize and bind to polypurine/polypirimidine regions in double-stranded helical DNA in a sequence-specific manner. These recognition rules are outlined by Maher III, L. J., et al., Science,1989;245:725-730; Moser, H. E., et al., Science,1987;238:645-630; Beal, P. A., et al, Science,1992;251:1360-1363; Cooney, M., et al., Science, 1988;241:456-459; and Hogan, M. E., et al., EP Publication 375408. Modification of the oligonucleotides, such as the introduction of intercalators and backbone substitutions, and optimization of binding conditions (pH and cation concentration) have aided in overcoming inherent obstacles to TFO activity such as charge repulsion and instability, and it was recently shown that synthetic oligonucleotides can be targeted to specific sequences (for a recent review see Seidman and Glazer, J Clin Invest 2003;112:487-94).
In general, the triplex-forming oligonucleotide has the sequence correspondence:













oligo
3′--A
G
G
T



 



duplex
5′--A
G
C
T



 



duplex
3′--T
C
G
A






However, it has been shown that the A-AT and G-GC triplets have the greatest triple helical stability (Reither and Jeltsch, BMC Biochem, 2002, September 12, Epub). The same authors have demonstrated that TFOs designed according to the A-AT and G-GC rule do not form non-specific triplexes, indicating that the triplex formation is indeed sequence specific.
Thus for any given sequence in the ApoB regulatory region a triplex forming sequence may be devised. Triplex-forming oligonucleotides preferably are at least 15, more preferably 25, still more preferably 30 or more nucleotides in length, up to 50 or 100 bp.
Transfection of cells (for example, via cationic liposomes) with TFOs, and formation of the triple helical structure with the target DNA induces steric and functional changes, blocking transcription initiation and elongation, allowing the introduction of desired sequence changes in the endogenous DNA and resulting in the specific downregulation of gene expression. Examples of such suppression of gene expression in cells treated with TFOs include knockout of episomal supFG1 and endogenous HPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res. 1999; 27:1176-81, and Puri, et al, J Biol Chem, 2001; 276:28991-98), and the sequence- and target specific downregulation of expression of the Ets2 transcription factor, important in prostate cancer etiology (Carbone, et al, Nucl Acid Res. 2003; 31:833-43), and the pro-inflammatory ICAM-1 gene (Besch et al, J Biol Chem, 2002; 277:32473-79). In addition, Vuyisich and Beal have recently shown that sequence specific TFOs can bind to dsRNA, inhibiting activity of dsRNA-dependent enzymes such as RNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res 2000; 28:2369-74).
Additionally, TFOs designed according to the abovementioned principles can induce directed mutagenesis capable of effecting DNA repair, thus providing both downregulation and upregulation of expression of endogenous genes (Seidman and Glazer, J Clin Invest 2003; 112:487-94). Detailed description of the design, synthesis and administration of effective TFOs can be found in U.S. Patent Application Nos. 2003 017068 and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 to Emanuele et al, and U.S. Pat. No. 5,721,138 to Lawn.
The agents of the present invention can be provided to the individual per se, or as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier.
As used herein a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
Herein the term “active ingredient” refers to the active agent (e.g. polypeptide, polynucleotide or antibody preparation), which is accountable for the biological effect.
Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. One of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979).
Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.
Suitable routes of administration may, for example, include oral, rectal, transmucosal, transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
Alternately, one may administer the preparation in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient's body.
Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmuc o s al administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The preparations described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
The preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975) “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1].
Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
Compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
Since the present inventors have shown that ApoB regulates VEGFR1 transcription, the present inventors contemplate identification of additional agents other that those listed herein above for the treatment of angiogenesis related disorders.
Thus, according to another aspect of the present invention, there is provided a method of identifying an agent which regulates angiogenesis comprising:
(a) introducing the agent into a cell;
(b) analyzing VEGFR1 transcription in the cell; and
(c) identifying the agent capable of regulating ApoB-dependent VEGFR1 transcription in the cell, thereby selecting the agent which regulates angiogenesis.
As used herein, the phrase “ApoB-dependent VEGFR1 transcription” refers to the transcription of VEGFR1 which requires the presence of a functional ApoB. The present inventors postulate that ApoB may up-regulate transcription either by binding directly to the promoter region or alternatively by binding to another polypeptide which is capable of binding to the VEGFR1promoter region.
Agents that are able to up-regulate ApoB dependent transcription of VEGFR1 include agents that increase the activity (i.e. transcriptional activity) or amount of endogenous ApoB and also agents that are able to mimic (i.e. compete with) ApoB's ability to enhance VEGFR1 transcription.
Any type of agent may be identified according to the method of the present invention, including but not limited to polynucleotide agents and polypeptide agents. Candidate agents encompass numerous chemical classes, such as organic molecules, e.g. small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents typically comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, pheromones, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
Candidate agents may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc., to produce structural analogs.
According to one embodiment, the agent that is capable of up-regulating ApoB dependent transcription is a peptide agent. An exemplary agent of the present invention is one that comprises a ApoB (i.e. a ApoB derived peptide).
The term “peptide” as used herein refers to a polymer of natural or synthetic amino acids, encompassing native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides even more stable while in a body or more capable of penetrating into cells.
Candidate peptide sequences may be screened by determining if there is an interaction between them and the VEGFR1 promoter. Exemplary methods for such screening include EMSA (electromobility shift assay) and chromatin precipitation. Such methods are known to one skilled in the art.
Alternatively or additionally, the candidate peptides may be screened for regulatory activity of VEGFR1 transcription. An exemplary method for analyzing such regulatory activity comprises transfecting a polynucleotide encoding the promoter region of VEGFR1 (i.e. an exogenous VEGFR1 promoter) linked to a detectable protein (i.e. reporter protein) into a cell—i.e. a reporter based assay. The method further comprises introducing the candidate peptide agents into the cell (e.g. by transfection of an expression vector encoding the agent) and detecting the detectable protein whereby the amount of the detectable protein reflects the transcriptional activity of the promoter. It will be appreciated that the polynucleotide sequence of any protein that may be readily detected may be transcriptionally linked to the VEGFR1 promoter. Thus for example, the protein may be a phosphorescent protein such as luciferase, a fluorescent protein such as green fluorescent protein, a chemiluminescent protein or may be a non-directly detectable protein for which an antibody is available for detection thereof. Cells for analyzing transcriptional activity are further described hereinbelow.
It will be appreciated that transcriptional activity of endogenous VEGFR1 may also be analyzed with VEGFR1 being detected using a detectable agent such as an antibody.
Once the minimal amino acid sequence of ApoB is identified that is capable of transcriptionally activating VEGFR1, other peptides may be synthesized (comprising conservative or non-conservative substitutions) in order to “tweak the system” and generate ApoB-derived peptides with improved characteristics i.e. comprising an enhanced transcriptional activity.
Another way of identifying agents that regulate angiogenesis is by performing a reporter assay wherein the 3′ untranslated region (UTR) (i.e. promoter) of ApoB is operatively attached to a reporter molecule and introduced into a cell together with the candidate agent, wherein a change in the amount of the reporter molecule is indicative of the agent which regulates angiogenesis.
Since the present inventors have shown that ApoB decreases angiogenesis, the present inventors further contemplate that analysis of the level of ApoB may be used to diagnose whether a cancer is metastatic or not. More specifically, the present inventors propose that a decrease in the level of ApoB compared to a control subject is indicative that the cancer is metastatic.
Thus, according to another aspect of the present invention there is provided a method of identifying a metastasized cancer in a subject in need thereof, comprising determining the level of ApoB in a fluid sample of the subject, wherein an up-regulation of the level of the ApoB compared to the level of ApoB in the fluid sample of a control subject, is indicative of a metastasized cancer.
Examples of fluid samples which may be analyzed for ApoB include, but are not limited to blood, plasma, saliva and urine.
The level of ApoB may be analyzed on the protein level (e.g. by using antibodies) or the polynucleotide level (i.e. RNA level).
Methods of Detecting the Expression Level of RNA
The expression level of ApoB RNA can be determined using methods known in the arts, including for example Northern Blot analysis, RT-PCR analysis, RNA in situ hybridization, in situ RT-PCR, DNA microarrays/DNA chips, oligonucleotide microarray.
Methods of Detecting Expression and/or Activity of Proteins
Expression and/or activity level of proteins expressed in the cells of the cultures of some embodiments of the invention can be determined using methods known in the arts including for example Enzyme linked immunosorbent assay (ELISA), Western blot, Radioimmunoassay, Fluorescence activated cell sorting (FACS), Immunohistochemical analysis, In situ activity assay and In vitro activity assays.
Comparison with control subjects may be effected on healthy subjects or cancer patients, wherein the cancer is known not to be metastatic.
Typically, the ApoB level is at least 1.5 times, at least two times, at least 4 times, at least 5 times the amount in control subjects in order to indicate metastasis.
As used herein the term “about” refers to ±10%.
The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique” by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
General Materials and Methods
Zebrafish Husbandry and Injection: Zebrafish were raised by standard methods. The Tg(fli1:EGFP)y1, Tg(fli1:nEGFP)y7 [Yaniv et al., Nat Med 12, 711-716 (2006)] and Tg(flt1:YFP)hu4624 [Hogan et al., Nat Genet 41, 396-398 (2009)] lines were previously derived. MOs and DNA were injected as further described below.
Cloning of mtp and Rescue of the Mutant Phenotype: The full-length coding sequence of zebrafish mtp was amplified by PCR from WT cDNA and cloned into a pCSDest vector [Villefranc et al., Dev Dyn 236, 3077-3087 (2007)] as further described herein below. mtp mRNA was synthesized with mMessage mMachine kit (Ambion) and injected at a concentration of 232 pg per embryo into the 1 cell stage cytoplasm or into the YSL at dome stage (approximately at 4 hpf).
Microangiography and Cell Transplants: 10 μM Atorvastatin (Sigma), 1 ug/μl DiI-LDL (Invitrogen), 6 μg/ml C6 in 0.5% BSA Fatty Acid Free (Sigma A8806) and Human Apolipoprotein B-100 (A50220H Meridian Life Science) in 0.5% BSA Fatty Acid Free (Sigma A8806) microangiography was performed at 2.5 dpf as described [Isogai et al., Dev Biol 230, 278-301 (2001)]. HEK293 cells co-transfected with Huh7MTP+apoB34 (see herein below) or un-transfected control cells were transplanted into the perivitelline space of one side of the yolk of 2.5 dpf stl; Tg(fli1:EGFP)y1 mutant or WT embryos, ventral to the developing SIVs [Nicoll et al., J Cell Mol Med (2008)].
In Situ Hybridization, Oil Red 0, and Alkaline Phosphatase (AP) Staining: Embryos were fixed overnight in 4% PFA and processed for AP staining [Habeck et al., Curr Biol 12, 1405-1412 (2002)], Oil Red 0 staining [Schlegel et al., Biochemistry 45, 15179-15187 (2006)], or whole-mount in situ hybridization using antisense mRNA probes for vegfr1, vegfr2, vegfr3 and VeCad as described [Pham et al., Dev Biol 303, 772-783 (2007)].
Zebrafish Western Blot: 3 dpf zebrafish embryos were processed for Western Blot as described [Yaniv et al., Nat Med 12, 711-716 (2006)]. VEGFR1 was detected using a 1:500 dilution of a chick polyclonal serum (Ayes Labs). Blots were then reblocked by standard protocols and probed with a 1:5000 dilution of α-tubulin antibody (Sigma).
Mice and Immunofluorescence: ApoE and LDLR null mice (Jackson Laboratory) and their WT C57B1/6 controls were handled according to the Weizmann Institute Animal Care and Use Committee. All the analyses were performed between 8-38 weeks (n=3 for each group) on mice fed with normal chow diet. Following anesthesia, aortas were dissected and frozen for further PCR analyses or fixed for 3 hours in 4% Para formaldehyde, equilibrated for 36 hours in 30% sucrose in PBS-T, embedded in OCT and frozen at −80° C. Slides of 7 μm sections were fixed in cold methanol, blocked with BSA and goat serum and stained overnight with anti-CD31 (BD 550274) and anti-VEGFR1 (Abcam AB2350) antibodies. Slides were then incubated for 2 hours with Cy3 anti-rabbit and Cy2 anti-rat antibodies (Jackson), stained with DAPI (10 mM Inno-Train Diagnostik) and mounted in fluorescent mounting medium. Images were captured using a Zeiss LSM780 confocal microscope.
Microscopy and Imaging: Zebrafish embryos used for imaging or in situ hybridization were treated with 0.00 3% phenylthiourea from 8hpf to inhibit melanin pigment formation. Confocal imaging was performed using a FVIOOO Olympus imaging system or Zeiss LSM780 upright confocal microscope equipped with ×20 NA 1.0 lens.
Injection of Zebrafish Embryos: The mtp [Schlegel, A. & Stainier, D. Y. Biochemistry 45, 15179-15187 (2006)], apoCII [Pickart, M. A., et al. PLoS One 1, e104 (2006);









vegfr1



(SEQ ID NO: 1)



5′-ATATCGAACATTCTCTTGGTCTTGC-3′, 



 



apoB



(SEQ ID NO: 2)



5′-CAACTTAGTGTCCATTTTTATCGGC-3′



and



 



apoAl



(SEQ ID NO: 3)



5′-TCAGTGCAAGAGCCACGAATTTCAT-3,






MOs (Gene-Tools) were resuspended and injected at concentrations from 5-20 ng/embryo.

pCS2vegfrlCDS mRNA (1200 ng/embryo) and pCS2mtpCDSmRNA (232 ng/embryo) were injected at the 1 cell stage or at dome stage, respectively.
Positional Cloning: Generation of map-cross lines, bulk segregant analyses, genomic DNA isolation and PCR were performed as described [Roman et al., Development 129, 3009-3019 (2002). Polymorphism analyses and sequence comparisons were performed using SeqMan alignment software (DNASTAR, Inc.).
Generation of pCS2mtpCDS and pCS2vegfr1CDS: The following primers were used to amplify the full-length coding sequences of zebrafish mtp and vegfr1:









mtp:



(SEQ ID NO: 4)



5′-ATGATGCCGGTTGCCGGACT-3′



and



 



(SEQ ID NO: 5)



5′-TTACCAGGCCGGCTCAAAGA-3′



 



vegfr1:



(SEQ ID NO: 6)



5′-GACCAAGAGAATGTTCGATATATTATTTGTG 3′ 



and



 



(SEQ ID NO: 7)



5′-TTAGAAACTGGGGTAAAGAAGATCGCCTTC-3′.






Following TOPO (Invitrogen) cloning and sequencing, a Gateway (Invitrogen) compatible middle Entry clone was generated by BP recombination. The mtp and vegfr1 coding sequences were then transferred into a pCSDest vector 5 using a Gateway LR reaction (Invitrogen) to give pCSmtpCDS and pCSvegfr1CDS. Following linearization with NotI, the vectors were used as templates for mRNA synthesis.
HEK293 Transfection: 50% confluent HEK293 cells were co-transfected with 1 μg each of ApoB34 and Huh7MTP for 6 hours, using fugene 6 (Roche). Following transfection, cells were labeled with Cell tracker orange CMRA (Invitrogen) for 45 minutes and grown overnight in serum supplemented DMEM media.
Cloning of Huh7MTP: Huh? (human hepatoma) cell RNA was subjected to RT-PCR using









(SEQ ID NO: 8)



5′-AGAAAGCTTGCTGGTCAATATGATTCTTCTTGC-3′



and



 



(SEQ ID NO: 9)



5′-AGATCTAGAATCACAGGTCAGTTTCAAAACCATCC-3′ 



 



primers.






The PCR product was cloned into pCMV5, sequenced and found to be identical to a human MTP cDNA (Locus EAX06106; accession CH471057.1). For functional characterization of Huh7MTP, see FIG. 14A-B.
Quantitative Analysis of HEK293 Cell Transplants: 3.5 dpf embryos, successfully transplanted with fluorescent red cells adjacent to green fluorescent endothelial cells were imaged and phenotypic quantitation was performed. The total length of the interface between endothelial and transplanted cells (see FIG. 15), as well as the total length of overlapping between the two cell populations (see FIG. 15) were traced and measured. The ratio r=L2/(L1+L2) was used to measure the ability of endothelial cells to migrate on top of ApoB secreting cells. The ratios were computed for three transplanted animals per treatment (transfected and un-transfected HEK293 cells), and were compared using t-test.
RNA Isolation from Mouse Aortic Tissue and RT-PCR Analyses: Dissected Aortas from C57B1/6 and ApoE null mice (500 mg tissue per sample) were homogenized in Trizol and processed for RNA isolation and semi-quantitative PCR. For all samples, cDNA was generated from equal amounts of RNA (600 ng). Primers to measure relative changes in vegfr1 mRNA transcripts were:









(SEQ ID NO: 10)



5′GGCCCGGGATATTTATAAGAAC-3′



and



 



(SEQ ID NO: 11)



5′-CCATCCATTTTAGGGGAAGTC-3′.






Expression levels were standardized to the primer set specific for β2-microglobulin:









(SEQ ID NO: 12)



5′-GTCTCGATCCCAGTAGACGG-3′



and



 



(SEQ ID NO: 13)



5′-TGGTGCTTGTCTCACTGACC-3′3′.






Quantitative analyses were performed using ImageJ.
Total RNA Isolation from Zebrafish Embryos, Semi Quantitative and Quantitative Real-Time PCR Analysis: 30-60 WT or mtp MO injected embryos were homogenized in Trizol (Invitrogen) and processed for RNA extraction following standard procedures. After precipitation, RNA was treated with DNase using the DNA free kit (Ambion) to eliminate genomic DNA.
1 μg of total RNA per reaction was reverse transcribed using the ThermoScript RT-PCR kit (Invitrogen). In yfp measurement experiments, RNeasy mini kit (QIAGENE) was used for RNA extraction and 1 μg of total RNA per reaction was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystem).
Primers to measure relative changes in mRNA transcripts were as follows:









vegfr1:



(SEQ ID NO: 14)



5′TGGTCATATGGAGTCCTGCTC-3′3′



and



 



(SEQ ID NO: 15)



5′-CATGTTGAGTCCTGGGTATGG-3′3′ 



 



vegfr2:



(SEQ ID NO: 16)



5′-CTGGTGGAGAGGCTAGGAGA-3′



and



 



(SEQ ID NO: 17)



5′-TGATCGGGATGTAGTGCTTTC-3′



 



vegfr3:



(SEQ ID NO: 18)



5′-TAACCAACCCCTCCATCAGA-3′



and



 



(SEQ ID NO: 19)



5′-CTGAATGCTGAGAGTCCGATT-3′ 



 



cdh5:



(SEQ ID NO: 20)



5′-GCACATGAAGATGTGTTGAATG-3′



and



 



(SEQ ID NO: 21)



5′-TGGTTAGTTCTGGTGCATTGTC-3′



 



fli1:



(SEQ ID NO: 22)



5′-CCATCTCACGGCTGACCAGT-3′



and



 



(SEQ ID NO: 23)



5′-GACAGCGCACACAACCAC-3′



 



yfp:



(SEQ ID NO: 24)



5′-CACATGAAGCAGCACGACTT-3′



and



 



(SEQ ID NO: 25)



5′-GGTCTTGTAGTTGCCGTCGT-3′.






Expression levels were standardized to the primer set specific for ef1α and β actin:









ef1a:



(SEQ ID NO: 26)



5′-CCTCTTTCTGTTACCTGGCAAA-3′



and



 



(SEQ ID NO: 27)



5′-CTTTTCCTTTCCCATGATTGA-3′



 



β actin:



(SEQ ID NO: 28)



5′-TGACAGGATGCAGAAGGAGA-3′



and



 



(SEQ ID NO: 29)



5′-GCCTCCGATCCAGACAGAGT-3′.






Reactions were run in 96-well plates in a LightCycler 480 Real-Time PCR System (Roche) or in Step One Plus real time PCR system (Applied Biosystem) and results were analyzed using built-in software. Measurements were conducted in duplicates.
For semi quantitative real-time PCR, primers used to measure relative changes in mtp mRNA transcripts were









(SEQ ID NO: 30)



5′-CCGTCTTACATGGAGGTGAA-3′



and



 



(SEQ ID NO: 31)



5′-CGGACATGGAGAACATCTTG-3.






Expression levels were standardized to the primer set specific for









β actin:



(SEQ ID NO: 32)



5′-CAGCTAGTGCGAATATCATCT-3′



and



(SEQ ID NO: 33)



5′-TTTCTGTCCCATACCAACC-3′.






Cell Cultures and Reagents: Human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) (VEC Technologies and Promo Cells) were cultured in complete MCDB 131 medium (VEC Technologies) or in M199 medium supplemented with ECGS (Zotal).
Boyden Chamber Assays: HUVECs or HAECs (50,000/well) were seeded on the top well of a Boyden Chamber (5 μm pore size) and cultured overnight at 37° C. and 5% CO2. Subsequently, the bottom wells were filled with either LDL(−) serum, or LDL(−) serum supplemented with 10 μg/ml LDL for overnight incubation. After incubation was completed, endothelial cells (ECs) that remained on the upper side of the membrane were scrapped off with a cotton swap. Endothelial cells that transmigrated were stained with DAPI and imaged using fluorescent microscopy. Positive nuclei were counted in 3 random image fields. Three individual experiments were conducted and the averages were used to generate the bar graph.
Wound Healing Assay: HUVECs exposed to either siRNA control (non-targeting) or siRNA for VEGFR1 were plated to confluency for wound assays. A 200 μm wound was inflicted in both groups and each was treated with either LDL or vehicle. Migration of cells was recorded for 24 hrs (note proliferation cycle of these cells ranges from 36 to 48 hrs). Four wells per experimental group were measured. Results were normalized to control and expressed as migration area over control.
Small Interfering RNA: siRNA reagents were obtained from Dharmacon Research, Inc (Chicago, Ill.). Endothelial cells at 90% confluency were transfected with DharmaFECT transfection reagents in the absence of antibiotics. For transfection either VEGFR1 siRNA SMARTpool or non-targeting siRNA (siCONTROL) were used at 100 nmol/L following manufacturer's instructions. Efficiency of the siRNA was evaluated by standard Western blots against VEGFR1 and resulted in 78 to 85% reduction of VEGFR1 protein. Experiments on migration were conducted 48 hrs post-transfection.
MTT Proliferation Assay: HUVECs were plated in 96 well plates (coated with 0.2% gelatin) at a density of 2000 cells/well in 0.2 ml growing medium. After 24 hours the medium was washed and cells were incubated with either 20% LPDS, or 20% LPDS supplemented with LDL 10 or 25 μg/ml for indicated periods. The number of cells was then determined using the MTT cell growth assay. Briefly, MTT reagent (Sigma M5655) was added to the wells at a final concentration of 0.6 mg/ml, the cells were further incubated at 37° C. for 2 hours. The reaction was terminated by adding 100 μl/well of an extraction solution consisting of 20% SDS in a 50% DMF solution. Absorbance was read at 570 nm using an ELISA plate reader, after leaving the plates protected from light overnight.
Fatty Acids Feeding of Zebrafish Embryos: Dechorionized 24 hours post fertilization (hpf) embryos were incubated with 6 μg/ml short, intermediate or long chain fatty acid in a solution containing 0.1% BSA Fatty Acid Free (Sigma A8806) for 48 hours. Embryos were then fixed overnight in 4% PFA and processed for AP staining.
Triglyceride Content Measurements: Dechorionized 24 hpf embryos were incubated with 2 μCi/ml 3H Oleic Acid (Perkin Elmer NET289005MC) in a solution containing 0.1% BSA Fatty Acid Free (Sigma A8806) and 3 μg/ml oleic acid (Sigma O1383) for 48 hours. After deyolking, the embryos were homogenized in 1 ml cold methanol. Lipids were extracted following the method of Bligh and Dyer [Can J Biochem Physiol 37, 911-917 (1959)] adding ice cold chloroform and DDW. The lower chloroform phase was collected and evaporated under vacuum or N2 and kept at −20° C. Lipids were developed on silica gel 60 TLC plates in a solvent system of Petroleum Ether: Diethyl Ether: Acetic Acid (80:20:1). Labeled lipids were visualized with autoradiography and spots corresponding to triglyceride standards (Sigma 17811) were scraped and quantified using liquid scintillation fluid. For HPLC, samples were lyophilized for 2 hours and 10 μl of pyridine and 80 μl of N-methyl-N-trimethylsilyl trifluoroacetamide were added to dry residue. The samples were then shaken vigorously for 30 seconds, and the mixture was transferred to a 2-ml autosampler glass vial with a 100-ml conical glass insert. After capping the vial, the reaction mixture was incubated at room temperature for at least 1 hour. For the GC-MS instrument and analytical parameters, CT split 1:25 mode was used. Cholesterol was identified by comparison of its retention time and mass spectrum to those generated for authentic standard analyzed in the same sequence. Relative quantification was performed using the reconstructed ion chromatogram generated from the characteristic cholesterol fragments: 329, 353, 368 and 458 Da.
FACS Sorting of ECs from Zebrafish Embryo: 3dpf Tg(fli1:EGFP)y1 embryos were used for FACS sorting of GFP labeled endothelial cells. Single cell suspensions were prepared as described in Takada et al [Dev Dyn 239, 2041-2047 (2010)]. Sorting was performed at 4° C. in FACSAria cell sorter using a 70 μm nozzle. GFP+ and GFP− cells were separately collected in 1 ml FCS. Sorted cells were washed with PBS and centrifuged at 300 g at 4° C. for 5 min twice. The cell pellet was suspended in 1 ml Trizol for RNA extraction.
HUVEC Immunoblotting: HUVECs were lysed for 30 minutes in mRIPA buffer. Proteins were separated by SDS-PAGE and transferred to nylon membranes (Whatman). Membranes were then probed with VEGFR2 (Cell Signaling) or VEGFR1 antibodies (R&D) and detected by enhanced chemiluminescence (Thermo Fisher Scientific).
Example 1
Stl, a Zebrafish Mutant Exhibiting Excessive Angiogenesis
The present inventors identified stalactite (stl), a zebrafish mutant displaying excessive sprouting angiogenesis, in a Tg(fli-EGFP)Y1 transgenic-based forward-genetic screen for vascular-specific mutations. stl mutants display ectopic angiogenic segments that extend ventrally from the subintestinal (SI) plexus (FIGS. 1A-D), a vascular bed that initially forms bilaterally over the dorsal-lateral aspect of the large zebrafish yolk cell (FIG. 5). Quantitative analysis (FIG. 6) reveals large increases in both the number and length of ventral SI sprouts in stl mutants (FIG. 1E) when compared to wild type siblings. Increased angiogenic branching is also observed in the dorsal part of the intersegmental vessels (ISVs) at slightly later stages of development (FIGS. 1F-H). Endothelial nuclei were counted in the yolk area of wild type and stl;Tg(fli:nEGFP)y7 embryos to determine whether formation of ectopic sprouts in stl mutants involved overproliferation of endothelial cells. Total SI endothelial cell number is increased by 42% in stl mutants, with excess cells found exclusively in the ectopic ventral sprouts (FIGS. 1I-J). stl mutants also display defects in yolk absorption (FIG. 5) and the larvae die by approximately 6 days post fertilization (dpf).
In order to examine the molecular nature of the stl phenotype, the present inventors positionally cloned the defective locus. The mutation was genetically mapped to an interval in linkage group 1 (See FIG. 7 for details) containing the gene encoding microsomal triglyceride transfer protein (mtp). Sequence analysis of mtp cDNA from stl mutants and their phenotypically wild type siblings revealed a change in a conserved leucine (Leu475) residue to a proline (FIG. 7). MTP, an intraluminal protein found within the endoplasmic reticulum of liver and intestine, is required for assembly and secretion of proatherogenic-, apoB-containing lipoproteins such as chylomicrons, very low-density lipoproteins (VLDLs), and low-density lipoproteins (LDLs). Following their assembly as mature particles, apoB-lipoproteins are secreted to the blood and lymph stream by MTP. Even though the MTP/apoB lipoprotein pathway was originally thought to be unique to the liver and intestine, the demonstration that the murine yolk sac expresses mtp and that mice homozygous for an mtp gene disruption die at E10.5 underscores the importance of the synthesis and secretion of apoB-containing lipoproteins during early embryogenesis. The yolk syncytial layer (YSL) of the fish (the functional counterpart of the yolk sac of higher vertebrates) expresses mtp starting at the gastrula stage and forms apoB-related lipoproteins, which enter the circulatory system and deliver nutrient lipids to the tissues. As in mice and humans, fish mtp is involved in lipoprotein assembly in the YSL and is found later in the intestine and liver. In addition, zebrafish have structural and functional homologues of mammalian apoAI, apoCII, apoE, phospholipase A2 and lipoprotein lipase.
Four additional lines of evidence confirm that a mutation in mtp is responsible for the stl phenotype. First, injection of antisense morpholino oligonucleotides (MOs) against mtp [Schlegel et al., Biochemistry 45, 15179-15187 (2006)], reproducibly phenocopies the angiogenesis and yolk absorption phenotypes seen in stl mutants (FIG. 1K and FIG. 8A). Second, injection of mtp mRNA into stl mutants suppresses ectopic sprouting and RNA-rescued larvae survive for at least 10 dpf (FIG. 11 and FIG. 8B). Third, downregulation of apoB using apoB MOs leads to ectopic SIV branching, reminiscent of that displayed by stl mutants (FIGS. 8C-E). This pro-angiogenic response however, is not observed upon down-regulation of apoA1 (FIG. 8F). Finally, reduction of LDL levels in WT embryos using atorvastatin, efficiently phenocopied the excess angiogenesis phenotype of stl mutants (FIG. 1M-0). Altogether these results confirm the specificity of the MTP/apoB pathway in causing excess angiogenesis.
The restricted expression of the mtp transcript in the yolk syncytium, liver, and intestine, and the lack of vascular expression (FIG. 8G), strongly suggests that its role in vessel development is non-cell autonomous. To confirm this notion, the present inventors injected mtp mRNA into the yolk syncytium at dome stage. As the cytoplasmic bridges between the embryo and the yolk cell are closed by the 1000-cell stage, reagents injected into the yolk cell after this stage are restricted exclusively to the yolk syncytium. As expected for a YSL-specific defect, injection of mtp mRNA at dome stage yielded efficient rescue of the stl excess angiogenesis phenotype (FIG. 8B).
In humans, mutations in mtp are the proximal cause of abetalipoproteinemia, an autosomal recessive defect in the production of apoB-containing lipoproteins that results in a virtual absence of LDL in blood. To confirm that the effects of mtp downregulation on lipoprotein production and/or secretion are conserved in zebrafish, WT and stl mutant embryos were stained with Oil Red O (ORO). As seen in FIG. 1P, Q, stl mutants display a clear absence of lipids in the vasculature (FIG. 1q) when compared to WT siblings (FIG. 1P). To further confirm the lipoprotein-depletion phenotype, a more detailed lipid analysis was performed. Taking advantage of the fact that exogenously supplied-, radioactively-labeled fatty acids are properly processed by zebrafish larvae, and are found as expected in the triglycerides (TGs) fraction, thin layer chromatography (TLC) was performed to confirm that triglycerides are strongly reduced in stl mutants (FIG. 9). Same results were obtained when the levels of cholesterol were analyzed using gas chromatography-mass spectrometry (GC-MS) (See FIG. 9 for details).
Example 2
The Excess Angiogenesis Phenotype is not Caused by Global Lipid Starvation
To further characterize the effects of lipoproteins on angiogenesis in vivo, the present inventors generated a zebrafish model of lipoprotein overload in circulation by down-regulating the apoCII gene. ApoCII is a component of apoB-containing lipoproteins required for activation of the enzyme lipoprotein lipase (LPL) in blood capillaries. LPL activation leads to triglyceride hydrolysis, and to release of free fatty acids for use by cells. In humans, mutations in apoCII result in accumulation of unprocessed lipoproteins in circulation (hyperlipoproteinemia type IB and hypertriglyceridemia), and increased risk of early atherosclerosis. The present inventors reasoned that while morpholino knockdown of apo CII early in development would lead to a global reduction in lipid delivery, the endothelial cells of these morphants would nevertheless be exposed to elevated concentrations of plasma lipoproteins, mimicking the hyperlipidemia described in higher vertebrates. Injection of apoCII MOs into Tg(fli-EGFP)y1 embryos did result in increased intravascular levels of lipoproteins as demonstrated by ORO staining at 4 dpf (FIG. 2A; compare with FIG. 1P,Q). This was accompanied by a significant reduction in TGs and cholesterol delivery to cells (FIG. 9B, C). In contrast to the excess angiogenesis phenotype displayed by stl mutants however, no ectopic SW sprouts were observed in apoCII (FIG. 2B-C) or lpl morphants (FIGS. 10A-D), confirming that global lipoprotein starvation per se does not lead to excessive angiogenesis. On the contrary, high lipoprotein levels in circulation resulted in a poorly developed vasculature characterized by short and partially lumenized ISVs (FIGS. 2D-F). These results are consistent with previous findings describing premature vascular disease [Connelly et al., J Clin Invest 80, 1597-1606 (1987)], and general endothelial dysfunction [Landmesser et al., Seminars in thrombosis and hemostasis 26, 529-537 (2000)] under hyperlipoproteinemic conditions in humans [Henry et al., The American journal of cardiology 72, 61C-64C (1993)].
To further demonstrate that the angiogenic phenotypes seen in stl and apoCII morphants result from a direct response of endothelial cells to lipoprotein levels, and not from alternative cues trigged by fatty acid depletion, the present inventors checked the ability of exogenously supplied fatty acids to restore a normal vasculature. Addition of short-, intermediate-, and long-chain fatty acids to the embryo media, did not rescue the vascular phenotypes in stl mutants (FIGS. 2G-K) or apoCII morphants (FIG. 2L, M, N and data not shown), even when applied at high concentrations (FIG. 11), confirming that the angiogenic phenotypes result from a direct response of endothelial cells to lipoprotein levels.
Example 3
Lipoprotein Levels Regulate VEGFR1 Expression
To elucidate the mechanisms underlying the effects of lipoproteins on vascular growth, the present inventors set out to identify the specific elements within endothelial cells on one hand, and apoB-lipoproteins on the other, responsible for the angiogenic phenotypes. They began by searching for endothelial specific candidates whose expression was affected by changes in lipoprotein levels. qRT-PCR (FIG. 3A) and in situ hybridization (FIG. 12) revealed dramatic reduction in vegfr1 mRNA in mtp MO-injected embryos at 24 hpf. This reduction was specific for vegfr1, since little change was detected in the levels of vegfr2, vegfr3, or other vascular genes (FIG. 3A, FIG. 12, and data not shown). VEGFR1 protein levels were also strongly reduced in stl mutants, but were significantly up-regulated in apoCII morphants, where endothelial cells face high concentrations of apoB-lipoproteins (FIG. 3B). Similar effects on VEGFR1 levels were observed in endothelial cells in vitro. Addition of LDL, the metabolic product of apoB-containing lipoproteins, to cultured HUVECs did not affect levels of VEGFR2, but elicited increases in VEGFR1 levels (FIG. 3C). Finally, hyperlipidemic apoE (FIGS. 3D-E, H) and LDL-R (FIGS. 3F-G) null mice also showed increased endothelial-specific VEGFR1 expression, confirming the conservation of this pathway among vertebrates.
Previous studies have shown that VEGFR1 plays an inhibitory role in angiogenesis, acting as a “sink” for VEGF ligand Hiratsuka, S., et al. Proceedings of the National Academy of Sciences of the United States of America 95, 9349-9354 (1998); Kearney, J. B., et al. Blood 99, 2397-2407 (2002); Chappell, J. C., et al., Developmental cell 17, 377-386 (2009). Downregulation of vegfr1 by ATG blocking morpholinos results in excess sprouting and branching from the SIV plexus, and trunk ISVs, reminiscent of those found in stl mutants (FIG. 13). The present inventors reasoned that if lipoprotein depletion promotes angiogenesis via down-regulation of VEGFR1, it should be possible to rescue the pro-angiogenic phenotype of stl mutants by vegfr1 overexpression. Indeed, injection of vegfr1 mRNA into stl mutant embryos suppresses ectopic SIV sprouting (FIGS. 3I-K). Furthermore, siRNA-mediated downregulation of VEGFR1 (FIG. 3L) abolished the ability of cultured endothelial cells to respond to LDL levels in a Wound Healing assay (FIG. 3M).
The changes in vegfr1 mRNA levels observed in stl mutants (FIG. 3A) and apoE null mice (FIG. 3H) could result from either transcriptional regulation, or post-transcriptional modifications that alter RNA stability of the vegfr1 transcript. To distinguish between these two possibilities, the present inventors took advantage of a transgenic zebrafish reporter expressing YFP under the regulation of the vegfr1 promoter, which efficiently recapitulates the pattern of expression of the endogenous vegfr1 gene. It was hypothesized that if lipoproteins regulate vegfr1 mRNA expression at the transcriptional level, downregulation of mtp will result in a significant decrease in the levels of yfp mRNA. If in turn, vegfr1 downregulation involves alterations in its mRNA stability, the levels of yfp mRNA will remain unchanged upon mtp MOs injection. As seen in FIGS. 3N-P, downregulation of the mtp/apoB pathway results in a significant decrease in the levels of the yfp transcript. These results provide evidence for a mechanism involving transcriptional regulation of vegfr1 in response to apoB-lipoprotein levels. Whether this is a direct or indirect regulation still remains to be elucidated.
Example 4
apoB Particles Regulate Angiogenesis by Directly Acting on Endothelial Cells
The present inventors next characterized the mechanism by which apoB lipoproteins exert their effects on angiogenesis. Since the vascular phenotype of stl mutants is not trigged by global lipid starvation (See FIG. 2), it was hypothesized that deficits of lipoproteins, directly sensed by endothelial cells, exert a pro-angiogenic response in these mutants. To test this hypothesis, the present inventors first examined whether exogenously supplied LDL could suppress ectopic sprouting in stl mutants when applied intravascularly. Injection of DiI-labeled LDL into 2.5 dpf stl; mutants resulted in strong reduction in both the number and length of ectopic SIV sprouts (FIGS. 4A-C), suggesting that circulating lipoproteins have the ability to activate pro- or anti-angiogenic mechanisms in endothelial cells.
To further confirm the cell autonomous nature of the endothelial response the present inventors designed an in vivo approach to allow for localized delivery of exogenous apoB-particles in the proximity of angiogenic vessels (FIG. 4D). Lipoprotein-secreting HEK293 cells co-transfected with vectors encoding human forms of mtp and apoB34 (FIG. 14) or untransfected HEK293 control cells were transplanted into the perivitelline space, on one side of 2.5 dpf stl;Tg(fli-EGFP)y1, animals, in close proximity to the forming SIVs (FIG. 4D). Lipoprotein-secreting cells impeded the migration of endothelial cells and the growth of ectopic sprouts in the area of transplantation (FIG. 4E, left panel). Ectopic SW sprouting however, was not affected on the untransplanted side of the same embryo (FIG. 4E, right panel), confirming that the localized transplantation did not result in systemic rescue of the stl phenotype. Non-lipoprotein-secreting cells, in contrast, did not impede sprouting or act as a barrier to endothelial migration (FIG. 4F). Quantitative analysis revealed a large decrease in endothelial migration onto lipoprotein-producing versus non-lipoprotein producing cells (FIG. 4G and FIG. 15). A similar direct inhibitory effect of LDL on endothelial cell migration was also observed in vitro (FIG. 16). The ability of ECs to migrate in Boyden chamber (FIG. 16A) and wound healing (FIG. 16B) assays was significantly impaired in LDL-supplemented media, even at concentration that did not affect cell proliferation (FIG. 16C). It is important to note however, that LDL levels considered pathological in human patients (>160 mg/dL), indeed result in endothelial cell death (32 and data not shown). Together the in vivo and in vitro results indicate that endothelial cells respond cell-autonomously to changes in apoB-lipoprotein levels. This ability is not restricted to their luminal surface, yet, it seems to require direct contact, as xenografts that were not placed in close proximity to the developing vessels, did not result in any inhibitory effect (data not shown).
Finally the present inventors asked which component—the lipid moieties or the apoB protein itself—within apoB-lipoproteins plays a role in eliciting an endothelial response. To answer this question the present inventors took advantage of the fact that stl mutants lack secreted apoB-lipoproteins, and attempted to rescue their vascular phenotype by restoring different elements of the complex (lipid vs. protein) individually. While intravascular supply of free fatty acids (FIG. 4H, I, K) did not rescue the ectopic branching phenotype of stl mutants, injection of a delipidated form of apoB-100, significantly reduced the number and length of ectopic angiogenic sprouts (FIG. 4H, J, K). These results strongly support the idea that the apoB protein itself, and not the individual lipid elements within apoB-lipoproteins, act on endothelial cells to directly regulate angiogenesis.
Endothelial-lipoprotein interactions have direct relevance to atherogenesis, thrombosis and other cardiovascular diseases. The results presented here reveal for the first time an intrinsic capacity of apoB-lipoproteins to control vascular growth in vivo, by regulating vegfr1 expression in endothelial cells (FIG. 4L).
Previous data hinted at direct interactions between the LDL and VEGF receptor families in vitro [Usui, R., EMBO Rep 8, 1155-1161 (2007); Yla-Herttuala, S. & Alitalo, K. EMBO Rep 8, 1127-1128 (2007), but the molecular mechanisms regulating this interplay remained obscure. The present examples show that VEGFR1 expression is regulated in vivo, by apoB-lipoprotein availability. Moreover it has been demonstrated that this regulation is mostly achieved at the transcriptional level.
Recent evidence suggests a potentially important role for the nutritional state of tissues in vessel growth. Taking advantage of the presently disclosed novel zebrafish models of hypo-, and hyperlipidemia, it has been possible to show that the effects of apoB-lipoproteins on angiogenesis are not trigged by reduced delivery of FA to tissues or global lipid starvation, as apoCII deficiency in zebrafish embryos did not phenocopy the vascular phenotype resulting from the loss of MTP. Further, the high plasma concentrations of TG-rich apoB-lipoproteins under these conditions inhibited angiogenesis, as did exogenous delivery of LDL. This effect however, required the presence of apoB, as intravascular supply of free FA did not affect vessel formation. Further supporting these findings, a delipidated form of apoB-100 efficiently reverted the excess angiogenesis phenotype of stl mutants, when applied intravascularly. These results support the notion that apoB itself provides the critical signal.
Several pathological conditions involve severe alterations in lipoprotein levels that result in a wide variety of vascular related diseases. These results have uncovered a novel mechanism of regulation of developmental angiogenesis by apoB-particles, which seems to be reactivated during pathological conditions of hyperlipidemia. These findings may provide novel explanations for endothelial dysfunction preceding the formation of atherogenic plaques, as well as for the impaired collateral vessel growth observed in hypercholesterolemic patients. Furthermore, these findings raise important questions about the potential effects of circulating lipoproteins during tumor-related angiogenesis.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

## Claims

1. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a recombinant apolipoprotein B (ApoB) polypeptide comprising SEQ ID NO: 34 and capable of transcriptionally upregulating VEGFR1-, thereby treating the cancer.

2. The method of claim 1, wherein said cancer is a metastatic cancer.

3. The method of claim 1, wherein said administering is effected in vivo.