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Matched Legal Cases: ['Application No. 60', 'Application No. 05806361', 'Application No. 743', 'Application No. 801', 'Application No. 2679', 'Application No. 2002', 'Application No. 2004', 'Application No. 2004', 'Application No. 2003', 'Application No. 202']

Patent US7579327 - Promoters exhibiting endothelial cell specificity and methods of using same - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsAn isolated polynucleotide functional as a promoter in eukaryotic cells is disclosed. The isolated polynucleotide includes an endothelial specific enhancer element as detailed herein. Further disclosed is a method of expressing a nucleic acid sequence of interest in endothelial cells....http://www.google.com/patents/US7579327?utm_source=gb-gplus-sharePatent US7579327 - Promoters exhibiting endothelial cell specificity and methods of using sameAdvanced Patent SearchPublication numberUS7579327 B2Publication typeGrantApplication numberUS 10/975,619Publication dateAug 25, 2009Filing dateOct 29, 2004Priority dateNov 17, 2000Fee statusPaidAlso published asUS20050112110, US20090326052, US20110201677, US20120201790, US20130296404Publication number10975619, 975619, US 7579327 B2, US 7579327B2, US-B2-7579327, US7579327 B2, US7579327B2InventorsDror Harats, Eyal Breitbart, Nira BloomOriginal AssigneeVascular Biogenics Ltd.Export CitationBiBTeX, EndNote, RefManPatent Citations (48), Non-Patent Citations (106), Referenced by (2), Classifications (38), Legal Events (3) External Links: USPTO, USPTO Assignment, EspacenetPromoters exhibiting endothelial cell specificity and methods of using sameUS 7579327 B2Abstract An isolated polynucleotide functional as a promoter in eukaryotic cells is disclosed. The isolated polynucleotide includes an endothelial specific enhancer element as detailed herein. Further disclosed is a method of expressing a nucleic acid sequence of interest in endothelial cells.
RELATED APPLICATIONS DATA This application is a Divisional of U.S. patent application Ser. No. 10/135,447, filed May 1, 2002, now U.S. Pat. No. 7,067,649, issued Jun. 27, 2006, which is a continuation-in-part of PCT/IL01/01059, filed Nov. 15, 2001, which claims priority from U.S. Provisional Patent Application No. 60/248,582, filed Nov. 17, 2000, the specifications of which are hereby incorporated by reference.
FIELD AND BACKGROUND OF THE INVENTION The present invention relates to isolated polynucleotide sequences exhibiting endothelial cell specific promoter activity, and methods of use thereof and, more particularly, to a modified-preproendothelin-1 (PPE-1) promoter which exhibits increased activity and specificity in endothelial cells. The invention further relates to modifications of the PPE promoter, which enhance its expression in response to physiological conditions including hypoxia and angiogenesis.
SUMMARY OF THE INVENTION According to one aspect of the present invention there is provided an isolated polynucleotide functional as a promoter in eukaryotic cells. The isolated polynucleotide includes an enhancer element including at least two copies of the sequence set forth in SEQ ID NO:6.
DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of an improved endothelial cell-specific promoter which can be employed to reliably direct high-level expression of a sequence of interest to endothelial cells and in particular endothelial cells participating in angiogenesis.
Example 1 Analysis of 3X-PPE-1 Plasmid Activity In-Vitro In order to analyze the activity of the PPE-1-3X, a comparison of reporter gene expression in the PPE-1-3X promoter plasmid and the unmodified PPE-1 promoter plasmid was undertaken. Reporter gene plasmids containing either the PPE-1-3X fragment or the unmodified PPE-1 fragment and the reporter gene Luciferase were transfected into endothelial and non-endothelial cell lines as well as to a bronchial epithelium cell line (B2B) which express the PPE-1 promoter (see materials and methods above). The B2B cell line was chosen to provide an indication of the 3X element's capacity to reduce expression in non-endothelial cell lines relative to the PPE-1 promoter. Transfection was accomplished using lipofectamine (Promega Corp., Madison, Wis.). A βgal-neo plasmid was employed as an indicator of the transfection efficiency in each case according to accepted molecular biology practice.
Example 2 Activity and Specificity of AdSPPE-1/Luciferase In-Vitro The PPE-1/Luciferase, PPE-1-3X/Luciferase, PPE-1/GFP and PPE-1-3X/GFP were also ligated into the Ad5 plasmid to produce Ad5PPE-1/Luc and Ad5PPE-1-3X/luc, Ad5PPE-1/GFP and Ad5PPE-1-3X/GFP (Varda-Bloom et al., (2001) Gene therapy 8:819-827). These constructs were assayed separately as detailed hereinbelow.
Example 3 Activity and Specificity of AdSPPE-3XLuc and Ad5PPE-3XGFP The Ad5PPE-3X/Luciferase and Ad5PPE-3X/GFP constructs were used to transfect the cell lines described hereinabove in Example 2 in order to ascertain the impact of the 3X element on specificity and expression levels. As in example 2, Ad5CMVLuc was used as a non-endothelial-specific control. Higher Luciferase expression in BAEC and HUVEC cell lines was detected under the control of the PPE-3X promoter as compared to the CMV promoter.
Example 4 In-Vitro Assay of Pro-Apoptotic Activity of the p55 Gene Following sub cloning of P55 (TNFR1, GenBank accession number M75866) into PACPPE3X (containing the PPE-1-3X promoter), and into PACCMV, co-transfection of these plasmids and GFP (pEGFP-C1 vector; CLONTECH, Palo Alto, Calif.). was performed as described hereinabove. Briefly, the gene was subcloned downstream to the PPE-1 promoter (instead of the luciferase gene) into the NotI restriction site, by T4 DNA ligase, following by transforming it into DH5α competent cells. Twenty four hours post-transfection, small and rounded apoptotic cells were visually discernible from normal cells. Electron microscopy of cells transfected with the pro-apoptotic plasmids showed typical appearance of apoptosis, confirming the visual evaluation.
Example 5 Hypoxia Responsive Element (HRE) Can Enhance Target Gene Expression in Hypoxic Sensitive Endothelial Cells Hypoxia is an important regulator of blood vessels' tone and structure. It has also been shown to be a potent stimulus of angiogenesis (in both ischemic heart diseases and cancer (Semenza, G. L. et al. (2000) Adv Exp Med Biol.; 475:123-30; Williams, K. J. (2001) Breast Cancer Res. 2001: 3;328-31 and Shimo, T. (2001) Cancer Lett. 174;57-64). Further, hypoxia has been reported to regulate the expression of many genes including erythropoietin, VEGF, glycolytic enzymes and ET-1. These genes are controlled by a common oxygen-sensing pathway, an inducible transcription complex termed hypoxia inducible factor-1 (HIF-1). The HIF-1 complex mediates transcriptional responses to hypoxia by binding the cis acting hypoxia responsive element (HRE) of target genes. The HRE is a conserved sequence located in the promoters of few genes that respond to hypoxia including: VEGF, Nitric Oxide Syntase-2, erytropoietin and others including endothelin-1, ET-1. The ET-1 promoter contains an inverted hypoxia response element at position −118 bp upstream of the transcription start site, the element contain 7 base pairs and is located between the GATA-2 and API sites 5′ GCACGTT 3′�50 base-pairs. (SEQ ID NO: 5.)
Example 6 Further Evaluation of PPE-1-3X and PPE-1 Promoter Activity in Endothelial Cell Lines FIG. 7 summarizes the results from B2B, HUVEC and BAEC transfection experiments using pPPE-1/Luciferase and pPPE-1-3X/Luciferase. Higher Luciferase expression (30, 8.5 and 1.5 times more) was observed under the control of the PPE-1-3X promoter than under the PPE-1 promoter in B2B, HUVEC and BAEC, respectively. These results confirm those presented hereinabove and serve to establish that PPE-1-3X is well suited to directing high level expression specifically to endothelial cells. In the context of future in-vivo delivery, the higher levels of expression achieved with the PPE-1-3X construct translate into administration of smaller amounts of DNA. This, in turn, will serve to increase specificity even further.
Example 7 Efficiency, Specificity and Stability of AdSPPE-1Luc In-Vivo In order to confirm that the endothelial specificity of expression observed in examples 2 through 6 was not an artifact of cell culture, the Ad5PPE-1/Luciferase construct was injected into C57BL/6 mice as described hereinabove in �Tissue gene expression in normal mice�. As in the in-vitro studies, Ad5CMV/Luciferase was employed as a negative control.
Example 8 Assays of Efficiency, Specificity and Stability of AdSPPE-1 In-Vivo�in BALB/C Mice The experiments of example 7 were repeated in 12 week old BALB/C mice (n=10 for each group) in order to demonstrate that the observed results were not an artifact of a particular strain of animals.
Example 9 Cellular Localization of Gene Delivered by AdSPPE-1 In-Vivo In order to ascertain cellular expression sites of the gene expressed by PPE-1 in-vivo, Green Fluorescent Protein (GFP) delivered by the adenoviral vector Ad5PPE-1-GFP was used. Ad5CMVGFP (Quantum, Canada) was used as non-endothelial-cell-specific negative control. Five days post-intravenous injection the mice were sacrificed and their tissues were analyzed by fluorescent microscopy.
Example 10 Assays of Efficiency and Endothelial Specificity of AdSPPE-1-3XLuc and AdSPPE-1-3X GFP In-Vitro
Example 11 Cellular Localization of a Reporter Gene Delivered by AdSPPE-1-3X In-Vivo In order to determine the cellular localization pattern of a reporter gene expressed under the control of the PPE-1-3X promoter in-vivo, Ad5PPE-1-3XGFP and Ad5PPE-1GFP were injected into mice as described hereinabove. Five days post-intravenous injection, the mice were sacrificed and their tissues were analyzed by a fluorescent microscopy.
Example 13 Assays of the Ad5PPE-1 Construct in a Carcinoma Cell Culture System In order to assay the efficiency of Ad5PPE-1 and Ad5CMV to drive Luciferase expression in cancerous cells, the D122-96 Lewis Lung Carcinoma cell line was employed.
Example 14 Assay of the Effect of the 3X Sequence in Tumor Angiogenic Blood Vessels In-Vivo In order to ascertain the effect of the 3X sequence on the PPE-1 promoter in angiogenic blood vessels, the Lewis Lung Carcinoma (LLC) metastases model (described hereinabove in material and methods) was employed. Five days post IV injection of 1010 infectious units of Ad5PPE-1GFP, Ad5PPE-1-3XGFP or Ad5CMVGFP, the mice were sacrificed and their tissues were analyzed as described in material and methods.
Example 15 Effect of the 3X Element on the PPE-1 Promoter in Tumor Angiogenic Blood Vessels In order to study the effect of the 3X element of the present invention on efficacy and specific activity of the PPE-1 promoter in tumor angiogenic blood vessels, the LLC metastases model was employed. Five days post i.v. injection of 1010 pfu/ml of Ad5PPE-1Luc, Ad5PPE-1-3XLuc, Ad5CMVLuc, Ad5PPE-1GFP, Ad5PPE-1-3X-GFP or Ad5CMVGFP, the mice were sacrificed and their tissues were analyzed for Luciferase or GFP expression as described hereinabove.
Example 16 Further Characterization of the PPE-1 Hypoxia Response In order to further characterize the effect of hypoxia on the murine PPE-1 promoter activity, bovine aortic endothelial cells (BAEC) were transfected by a DNA plasmid (pEL8; FIG. 26A). The pEL8 plasmid contains the murine PPE-1 promoter (1.4 kb) (red), the luciferase gene (1842 bp), the SV40 poly A sites and the first intron of the endothelin-1 gene, all termed the PPE-1 promoter cassette was digested and extracted by BamHI restriction enzyme as described in material and methods. Following transfection, cells were subjected to hypoxic conditions.
Example 17 Effect of the 3X Sequence on the PPE-1 Hypoxia Response In order to ascertain the effect of the 3X sequence on the PPE-1 hypoxia response, BAEC were transduced by Ad5PPE-1Luc and Ad5PPE-1(3X)Luc. Following transduction, the BAEC cells were incubated either in a hypoxic or a normoxic environment as detailed hereinabove. Results are summarized in FIG. 24. Luciferase expression using the Ad5PPE-1Luc construct significantly increased (seven folds) in response to hypoxia (2578 in hypoxia and 322.1 in normoxia). In contrast, the Ad5PPE-1(3X)Luc construct exhibited only 1.5 fold increase in response to hypoxia (from 2874.5 in normoxia to 4315 in hypoxia conditions). These results indicate that the high normoxic level of expression observed when the 3X sequence is added to the PPE-1 promoter serves to mask the hypoxic response to some extent.
Example 18 Assays of the PPE-1 Response to Hypoxia in a Transgenic Mouse Model In order to examine the murine PPE-1 promoter activity in tissues subjected to regional hypoxia/ischemia, mPPE-1-Luc transgenic mice, described hereinabove in materials and methods, were employed. The mice were induced to regional hind limb ischemia as previously described (Couffinhal T. et al. (1998) Am. J. Pathol. 152; 1667-1679). In brief, animals were anesthetized with pentobarbital sodium (40 mg/kg, IP). Unilateral ischemia of the hind limb was induced by ligation of the right femoral artery, approx. 2 mm proximal to the bifurcation of the saphenous and popliteal arteries. To verify the induction of functional change in perfusion, ultrasonic imaging was performed on days 4 and 14 by Synergy ultrasound system (GE) equipped with a 7.5 MHz transducer and angiographic software. Animals were housed under conventional conditions for up to 18 days.
Example 19 Effect of Level of Cellular Proliferation on AdSPPE-1Luc Activity in Endothelial Cells In order to ascertain the effect of level of cellular proliferation on efficiency and specific activity of Ad5PPE-1Luc, an angiogenic model of endothelial cells (BAEC), was tested in-vitro. Transduced BAEC were either induced to quiescence by serum deprivation or grown in 10% FCS for normal proliferation. Briefly, cells were transduced for 48 hours either as quiescent cells�72 hours post serum deprivation or as proliferating cells�in normal media (10% FCS). Luciferase activity is expressed as light unit/μg protein, to normalize for the difference in cell amount. The results presented are an average of triplicate test from four representative independent experiments.
Example 20 Assays of the PPE-1 Promoter in Atherosclerosis Induced Mice In order to test the efficiency and specificity of the Ad5PPE-1 vector in atherosclerotic blood vessels, 1010 pfu/ml of the viral vectors were systemically injected to 6 month old ApoE deficient mice (Plump, A. S. et al. Cell; 1991; 71:343-353).
Example 21 Assays of the PPE-1 Promoter in a Wound Healing Model In order to test the Ad5PPE-1 constructs efficiency and specific activity in directing Luciferase expression to healing wound blood vessels, a murine wound healing as described hereinabove in Material and Methods was employed.
Example 22 Targeted Expression of VEGF and PDGF-B to Ischemic Muscle Vessels In-vivo induction of angiogenesis oftentimes results in a primitive vessel network consisting of endothelial cells. These nascent vessels rupture easily, prone to regression and leakiness and poorly perfused. To overcome these limitations localized, timed and dose-controlled delivery of various angiogenic factors, capable of recruiting endothelial cells as well as periendothelial cells (i.e., pericytes in small vessels or smooth muscle cells in larger vessels) is desired.
Example 24 Prolonged Effect of VEGF Gene Therapy by PPE-Regulated Expression Tissue specific expression versus constitutive expression of pro-angiogenic factors was addressed with respect to the induction of angiogenesis. The effects of PPE-regulated and CMV-regulated VEGF expression on perfusion and angiogenesis were tested in 70 days long experiments. Mice with ischemic limb were treated as above (see Example 23). US imaging revealed significant improvement in perfusion in both treatment groups beginning 1-2 weeks following virus administration, while minor changes were detected in the control group (data not shown). The long-term effect of the Ad5PPE-1-3XVEGF treatment was detected 50 and 60 days following femoral artery ligation. Perfusion was significantly increased in the Ad5PPE-1-3XVEGF treated mice, as compared to Ad5CMVVEGF or saline-treated mice. The difference in perfusion between Ad5CMVVEGF and control treated animals decreased over that time interval. On the 50th day, mean intensity of perfusion in the Ad5PPE-1-3XVEGF treated group was about 50% higher than in the Ad5CMVVEGF or saline treated mice, and similar to that of the contralateral normal limb (p<0.01, FIG. 44A). Upon sacrifice of the animals on the 70th day, the capillary density in the muscle sections of Ad5PPE-1-3XVEGF treated mice was 747 CD31+ cells/mm2, which is 57% and 117% higher than in the Ad5CMVVEGF (474 CD31+cells/mm2) and control (342 CD31+ cells/mm2) groups, respectively (p<0.01, FIG. 44B).
Example 25 Enhanced Angiogenesis by PPE-Promoter Endothelial-Specific PDGF-B Expression PDGF-B is a paracrine endothelial secreted factor, which has been shown to be involved in vessel maturation by recruitment of smooth muscle cells, and probably also in angiogenesis [Edelberg, J. M. et al. Circulation 105, 608-13. (2002); Hsu et al. J Cell Physiol 165, 239-45. (1995); Koyama, N. et al. J Cell Physiol 158, 1-6. (1994)]. It has also been shown that PDGF-B is involved in intimal thickening [Sano, H. et al. Circulation 103, 2955-60. (2001); Kaiser, M., et al. Arthritis Rheum 41, 623-33. (1998)] and in fibroblast proliferation [Nesbit, M. et al. Lab Invest 81, 1263-74. (2001); Kim, W. J. et al. Invest Ophthalmol Vis Sci 40, 1364-72. (1999).]. The ability of PDGF-B to induce angiogenesis under endothelial specific regulation was tested in vitro and in-vivo.
Example 26 Vessel Maturation by PDGF-B Expression in Endothlelial Cells The assumption that further enhancement of angiogenesis and maturation of vasculature can be achieved by utilizing both VEGF and PDGF-B in a combination therapy was tested using two modalities of treatment: (i) single administration of 109 PFUs of Ad5PPE-1-3XVEGF and of Ad5PPE-1-3XPDGF-B; (ii) administration of similar dose of Ad5PPE-1-3XPDGF-B 5 days following administration of Ad5PPE-1-3XVEGF. Both modalities yielded the same results, and therefore are referred to as one. 90 days following ligation, both the combination therapy and the Ad5PPE-1-3XVEGF treated mice exhibited significantly higher capillary density as compared to the control, Ad5PPE-1-3XGFP treated mice, but there was no significant difference among the various therapeutic groups (FIG. 46B). However, the mean intensity of perfusion in US imaging in the combination therapy group was up to 42% higher than the Ad5PPE-1-3XVEGF treated group (FIG. 46A). This can be explained by maturation of small vessels in the ischemic muscles of the combination therapy groups and Ad5PPE-1-3XPDGF-B treated mice. Significant staining for vascular smooth muscle cells was seen in muscle sections from mice treated with the combination therapy or Ad5PPE-1-3XPDGF-B, immunostained for α-SMactin (FIGS. 46C-D). Sparse staining could be seen in control and Ad5PPE-1-3XVEGF treated mice (FIGS. 46E-F). In the normal limb muscles there was prominent staining around larger arterioles and venules (FIG. 46G). Similar results were obtained as early as 35 days following ligation in mice treated with Ad5PPE-1-3XPDGF-B (data not shown). No chronic changes were apparent in liver sections of treated mice 35 days following ligation.
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"Salvage of Infarcted Myocardium by Angiogenic Action of Basic Fibroblast Growth Factor", Science, New Series, 257(5075): 1401-1403, 1992.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS8293231 *Aug 13, 2010Oct 23, 2012Blue Blood Biotech Corp.Methods and compositions for treating ischemiaUS20100303800 *Aug 13, 2010Dec 2, 2010Blue Blood Biotech Corp.Methods and compositions for treating ischemia* Cited by examinerClassifications U.S. Classification514/44.00R, 435/320.1, 536/24.1International ClassificationA61P17/00, C12N5/10, A61K45/06, C12N15/09, A61P43/00, A61P9/00, A61K31/7088, A61P35/00, C12N15/63, C07K14/575, A61K48/00, C07K14/475, C07K14/515, C12N15/85, C07H21/04Cooperative ClassificationA61K48/0058, C07K14/57536, C12N2830/42, C12N15/113, C07K14/515, C12N2840/445, C12N2830/008, C12N2830/15, A61K48/00, C12N2830/85, C12N15/86, C12N2830/002, C07K14/475, C12N2830/001, C12N15/85European ClassificationA61K48/00D2, C07K14/515, C07K14/475, C07K14/575G, C12N15/85Legal EventsDateCodeEventDescriptionFeb 14, 2013FPAYFee paymentYear of fee payment: 4Nov 24, 2008ASAssignmentOwner name: VASCULAR BIOGENICS LTD., ISRAELFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BLOOM, NIRA;REEL/FRAME:021880/0558Effective date: 20081105May 5, 2008ASAssignmentOwner name: VASCULAR BIOGENICS LTD., ISRAELFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BREITBART, EYAL;REEL/FRAME:020896/0751Effective date: 20080212RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google