CA2200846C - Promoter for the receptor tyrosine kinase, tie - Google Patents

Abstract

The present application discloses promoter sequences for Tie, an endothelial cell receptor tyrosine kinase and their use in therapy and diagnosis as well as production of proteins in blood and tissues.

Description

PROMOTER FOR THE RECEPTOR TYROSINE RINASE, TIE
FIEhD OF THE INVENTION
The present invention relates generally to receptor tyrosine kinases and promoters thereof.
BACKGROUND OF THE INVENTION
The circulatory system is the first organ system to differentiate in the developing embryo.
Kaufman, The Atlas of Mouse Development, Academic Press, (1992). Embryonic and yolk sac vascular systems take form in an 8.5 day p.c. mouse embryo and a day later the heart beats regularly, circulating primitive blood cells, nutrients, and metabolic waste products. Endothelial cells covering blood vessels provide a barrier between blood and other tissues of the embryo. When organs differentiate and begin to perform their specific functions, the phenotypic heterogeneity of endothelial cells increases. Fenestrated vessels, nonfenestrated vessels with tight junctions and sinusoidal vessels are found, for example, in the kidney, brain, and liver, respectively. In addition, endothelial cells perform specific functions in differentiated tissue. For example, such cells take part in several biochemical and physiological events such as blood cell trafficking, blood clotting, hemostasis, ovulation, wound healing, atherosclerosis, and angiogenesis associated with tumor metastasis.
At least five receptor tyrosine kinase genes are expressed in endothelial cells. Of these, the protein products of the FLT1, KDR/FLK-1, and FLT4 genes belong to receptor tyrosine kinase subclass III; whereas Tie and its close relative Tek SUBSTITUTE SNEET RULE 26) WO 96/09381 P~.'TIFI951GOSIa ~IvIN~~

(Tie-2) form a novel subclass of their own (Terman, et al., Oncogene, 6: 1677-1683, 1991, Terman, et al., Biochem. Biophys. Res. Comm., 187: 1579-1586, 1992, Aprelikova, et al., Cancer Res., 52: 746-748, 1992, De Vries, et al., Science, 255: 989-991, 1992, Pajusola, et al., Cancer Res., S2: 5738-5742, 1992, Sarzani, et al., Biochem. Biophys. Res. Comm., 186:
706-714, 1992, Galland, et al., Oncogene, 8:
1233-1240, 1993, Millauer, et al., Cell, 72:
835-846', 1993, Oelrichs, et al., Oncogene, 8: 11-18, 1993, Schnurch and Risau, Development, 119: 957-968, 1993). Both human and mouse Tie cDNAs have been cloned (Partanen, et al., Mol. Cel. Biol., 12:
1698-1707,1992, Korhonen, et al., Blood, 80:
2548-2555, 1992, Korhonen, et al., Oncogene, 8:
395-403, 1994, Iwama, et al., Biochem. Biophys. Res.
Comm., 195: 301-309, 1993, Sato, et al., Proc. Natl.
Acad. Sci. USA., 90: 9355-9358, ,1993). Tie and homologous genes have been isolated from bovine and rat sources (Maisonpierre, et al., Oncogene, 8:
1631-1637,1993, Sato, et al., Proc. Natl. Acad. Sci.
USA., 90: 9355-9358,:1993). Genomic clones for mouse Tie and both mouse and human Tie promoter regions have been cloned and characterized.
The 4.4 kb Tie-encoding mRNA encodes a 125 kDa transmembrane protein which is N-glycosylated.
In its extracellular domain Tie contains two immunoglobulin-like loops and three epidermal growth factor and fibronectin type III homology regions, which are followed by traps- and juxtamembrane domains connected to a tyrosine kinase domain which is split by a short kinase insert sequence and a carboxyl terminal tail (Partanen, et al., Mol. CeI.
Biol., 12: 1698-1707, 1992, Korhonen, et al., Oncogene, 8: 395-403, 1994, Sato, et al., Proc.
a SItgSTITUTE SHEET (RULE 26) WO 96/09381 ~ ~ ~ 4 ~ PCT/FI95100520 Natl. Acad. Sci. USA., 90: 9355-9358, 1993). Both Tie and TEK have been localized to mouse chromosome 4 at a distance of 12.2 cm from each other. Such receptors are uniformly expressed in endothelial cells of various blood vessels during embryonic development, although the expression of Tek mRNA
appears to begin 0.5 days earlier than the expression of Tie. In adult mice, the expression of Tie mRNA persists in vessels of the lung whereas in the heart and brain it appears to decrease.
Korhonen, et al., Oncogene, 8: 395-403, (1994).
Production of Tie mRNA is enhanced during ovulation and wound healing and in human glioblastomas (Korhonen, et al., Blood, 80: 2548-2555, 1992).
Endothelial cells play a key role in gene therapy directed to diseases involving endothelial cells and blood vessels, such as establishment of neovascularization or inhibition of angiogenesis, and control of inflammatory trafficking of leukocytes. One approach to the treatment of vascular disease is to express genes at specific sites in the circulation that might ameliorate the disease in situ. Because endothelial cells are found at diseased sites, they represent logical carriers to convey therapeutic agents that might include anticoagulant, vasodilator, angiogenic or growth factors. Accordingly, the genetic modification of endothelial cells represents a therapeutic approach to the treatment of many vascular disorders, including hypertension, atherosclerosis and restenosis. For example, endothelial cells expressing growth inhibitory ' proteins could be introduced via catheter to the angioplasty site to prevent local intimal hyperplasia and clinical restenosis. The luminal SUBSTITUTE SHEET (RULE 26~

WO 96109381 ~ PC.TIFi95/0U52U . .

surface of vascular grafts could also be lined with genetically modified endothelial cells producing therapeutic proteins which prevent thrombosis or promote repooulation (Nabel, et al., J. Am. Coll.
Cardiol., 17, 189B-194B, 1991).
Endothelial cells lining blood vessels are easily transfected with methods using liposomes, adenovirus vectors and retroviral vectors (Nabel, et al., J. Am. Coll. Cardiol. 17: 189B-94B).
l0 Endothelial cells are also in direct contact with blood and are therefore optimal sources for production and secretion of desired proteins or peptides into the blood stream. For example, the Factor VIII gene may be introduced into endothelial cells under an endothelial cell- specific promoter, resulting in correction of hemophilia if the protein were expressed in sufficeint quantity. on the other hand, endothelial cells are also useful for delivery of peptides or proteins expressed in them into tissues. In this regard, a selective expression of a particular gene regulatory element in endothelial cells of the microvasculature (capil.laries) is extremely useful; given that most of the cell surface area facing the vascular lumen consists of microvascular endothelial cells.
Control elements of the endothelial cell specific promoters may be further subdivided and dissected into functional elements and units according to methods standard in the art. The Tie 3o protein is expressed in certain endothelial cells and about 0.9% of human bone marrow cells.
Therefore, it is likely that the Tie promoter is active also in some hematopoietic cells. However, expression of the Tie promoter in hematopoietic cells may be controlled by elements which are f SUBSTITUTE SNEET (RULE 26~

WO 96/09381 ~ P'~.'T/FI95/005~0' distinguishable from endothelial- cell-specific elements and may be dissected away while retaining the endothelial cell specificity of the promoter.
The present invention provides a novel promoter associated with the gene encoding the Tie receptor tyrosine kinase for use in therapeutic and diagnostic procedures. In addition, the promoter may prove useful in the production of desired proteins to the blood or tissues of animals.
SUMMARY OF THE INVENTION
The present invention generally relates to promoter sequences for the receptor tyrosine kinase, Tie. In a preferred embodiment of the invention, a mouse Tie promoter is provided comprising the sequence shown in SEQ ID NO:1. Also in a preferred embodiment, a human Tie promoter is provided comprising the sequence shown in SEQ ID N0:2. A
promoter according to the invention drives the expression of endothelial cell receptor tyrosine kinases, and in particular, the receptor tyrosine kinase, Tie.
~ vector according to the present invention may be any vector suitable for incorporating a promoter according to the invention , and may preferably be the 0.73mTIEpromGL2 vector deposited on September 19, 1994 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, as Accession Number 75892 Host cells according to the invention :nay be any host cell capable of housing the promoter or a vector containing the promoter according to the invention. Examples of host cells according to the invention are LEII endothelial cells.

SUBSTITUTE SHEET (RULE 26) WO 96/09381 , ~ ' PC'T/F79510~S2u . ~ , .

Other advantages and uses of the invention will be apparent upon consideration of the following Detailed Description thereof.
DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic diagram of the mouse Tie gene and promoter.
Figure 2 is a comparison of mouse and human Tie promoter sequences. (SEQ ID NO:1 and SEQ ID N0:2).
Figure 3 shows results of an analysis of Tie promoter activity.
Figures 4A shows expression patterns of the Tie promoter in the developing endocardium and_ head mesenchyme of 8.5 day mouse embryos.
Figure 4B show expression of a mouse Tie promoter construct in yolk sac blood islands in 8.5 day embryos.
Figure 5A and 5B show the expression pattern of mouse Tie promoter in 9.5 day embryos.
Figures 5C and 5D show the expression pattern of mouse Tie promoter in 11.5 day embryos.
Figure 6A shows expression of the Tie promoter in-9.5 day embryonic heart tissue.
Figure 6B shows expression of the Tie promoter in 11.5 day embryonic lung tissue.
; Figure 6C shows expression of the Tie promoter in 15.5 day embryonic brain tissue.
Figure 6D shows expression of the Tie promoter in 15.5 day embryonic liver tissue.
Figure 6E shows expression of the Tie promoter in developing bone trabeculae.
Figure 6F shows expression of the Tie promoter in developing kidney tissue.
SUSST1TUTE SHEET (RULE 26) WO 96/09381 ~ ~ ~ ~ ~ PGT/FI95I00520 -Figure 7A shows expression of the Tie promoter in the interalveolar capillaries of the lung in an 8-week-old mouse.
Figure 7B shows expression of the Tie promoter in the endothelial network of the bone marrow in an 8-week-old mouse.
Figure 7C shows expression of the Tie promoter in kidney tissue of an 8-week-old mouse.
Figure 7D shows expression of the Tie promoter in heart tissue of an 8-week-old mouse.
Figure 7E shows expression of the Tie promoter in liver tissue of an 8-week-old mouse.
Figure 7F shows expression of the Tie promoter in brain tissue of an 8-week-old mouse.
DETAINED DESCRIPTION OF THE INVENTION
The present invention provides promoter sequences capable of directing the expression of recombinant DNA sequences in endothelial cells. In particular, the invention provides promoter sequences which direct expression of the beta-galactosidase reporter gene in endothelial cells of mouse tissues. Promoters for production of proteins and peptides which act as anticoagulants, vasodilator inhibitors of thrombosis or restenosis into endothelial cells, blood and tissues.
Promoters according to the present invention are useful for directing expression of proteins and peptides for human gene therapy, antigens and markers useful for endothelial cell tagging, and antisense RNA constructs for use in endothelial cells in vivo and in vitro. Promoters, vectors, and host cells according to the invention are also useful in gene therapy for promoting expression of various growth factors or receptors or their SUBSTITUTE SHEET (RULE 26) ~p~~46 WO 96/09381 PCT/F'I95100520 _ g domains. Moreover, analogs of promoters according to the invention are useful for inhibiting undesired endothelial cell proliferation as, for example, the inhibition of angiogenesis during tumor formation.
EXAMPLE I
Cloning and characterization of the genomic Tie DNAs.
A. Mouse Genomic Tie In order to characterize the genomic organization of the mouse Tie gene, approximately 3 x 106 plaques were screened. The plaques were obtained from a genomic library made from DNA of adult SV129 mouse liver cells (Clontech) using as a probe a mouse 1C1D cDNA fragment (Korhonen, et al., Blood., 80:2548-2555, 1992) encoding the epidermal growth factor homology domains [GCVKDCPGCLHGGVCHDHDGCVCPPGFTGTRCEQACREGRFGQSCQEQCPG
TAGCRGLTFCLPDPYGCSCGSGWRGSQCQEACAPDHFGADCRLQCQCQNGGT
CDRFSGCVCPSGWHGVHCEKSDRIPQIL: SEQ ID N0:3] Three separate clones, SV1, SV2, and mTie were obtained thereby and each was subcloned into pGEM 3Zf(+) (Promega) and characterized by partial dideoxy chain termination sequencing and restriction enzyme analysis. A schematic structure of the mouse Tie gene and its promoter is shown in Figure 1. In that Figure, the positions of introns are indicated by arrows and their lengths are indicated. Restriction mapping, PCR, and nucleotide sequence analysis showed that the Tie gene spans approximately 19 kb of genomic DNA. Tie is encoded by 23 exons. The distinct structural domains of the,extracellular portion are encoded by either one exon each, comprising the first immunoglobulin-like loop, epidermal growth factor homology domains 1-3 and SUBSTITUTE SHEET (RULE 26j zzaas~s : :..
. . .
WO 96/09381 . ~ Pt,'T1FI9516051b.
- g _ f ibronectin-like domains 2 and 3, or by two exons comprising the second immunoglobulin-like loop and first fibronectin-like domain. The transmembrane region is encoded by a distinct exon; whereas the tyrosine kinase domain containing the kinase insert is encoded by eight exons of which the first encodes the juxtam~mbrane region. The lengths of the introns vary from 80 by to 2.6 kb.
H. Human Genomic Tie Three human Tie clones were isolated from a human placental genomic DNA library in the EI~L-3 vector system (Clontech) as shown in Partanen, et al., Mol.
Ce~l. Eiol., 12: 1698-1707 (1992), To obtain the human Tie clones, a PCR fragment encoding the Tie signal sequence was amplified from human Tie cDNA using the primers, 5'-CCCACATGAGAAGCC-3' (SEQ ID NO:. 4) and 5'-TGAGATCTTGGaGTATGGTCTGGCGGGTGCCC-3' (SEQ ID NQ:
5), and used to probe the aforementioned library.
The resulting positive clone containing the longest insert was plaque-purified and an approximately 7 kb SacI fragment was subcloned in pGEM 3Zf(+) and characterized. The resulting human Tie promoter sequence is shown in Figure 2. In that Figure, ? transcription initiation sites are marked with an asterisk (See primer extension and RNAse protection experiments below). Restriction endonuclease cleavage sites discussed herein are marked in bold.
A comparison of the genomic DNA sequences of mouse and human Tie promoters is also shown in Figure z. In that Figure, the mouse sequence extends from the 3' end of the first~exan to the AfZII site which is approximately 821 by upstream a SUBSTITUTE SHEEP (RULE 26) .~ .-'- ,'- y WO 96/09381 , ' ,PCT/FI95/~52~

from the ATG cvdon. A CA repeat found only in the mouse sequence is highlighted in bold in Figure 2.
EXAMPLE II
Determination of The Transcription Initiation Site in The Human Tie Gene For primer extension analysis of Tie-encoding nucleic acids, primer was labelled according to the manufacturer's instruction (Promega, USA). An aliquot of l0 pmol primer was then incubated with 10 x forward exchange buffer (Promega) , 10 ~,Ci/ml ['y-3zP] -ATP, and l0U T4 polynucleotide kinase at 37°C for 1 hour. The kinase was then inactivated by heating at 90°C for 2 minutes and the labelled primer was ethanol precipitated.
Poly (A+) RNA (20 ~,g) and 5 x 105 cpm labelled primer were then annealed in hybridization buffer (40 mM PIPES pH 6.4, 1mM EDTA pH 8.0, 0.4 M
NaCl and 80% formamide) by heating at 95°C for 12 minutes. Samples were then cooled slowly and ethanol precipitated. The resulting dried annealing mixture was suspended in primer extension buffer (SO mM
Tris- HC1, 50 mM KC1, 10 mM MgCl2, 10 mM DTT, 2 mM
each of deoxy ATP, deoxy CTP, deoxy GTP, and deoxy ~ TTP, 0.5 mM spermidine, pH 8.3, at 42°C) and 20 U
RNAsin and 40 U AMV reverse transcriptase were added. After 2 hours of incubation, template RNA was digested by addition of 20 ~g/ml RNAse A in 100 mM
NaCl, 10 mM Tris-HC1, 1 mM EDTA, pH 7.4, 37°C for 15 minutes. The resulting mixture was phenol extracted and ethanol precipitated. The pellet was then resuspended in loading dye (980 formamide, 10 mM
EDTA, O.lo xylene cyanol, 1% bromophenol blue) and loaded onto a 9% polyacrylamide/7M urea gel. After p f SUBSTITUTE SHEET (RULE 26) ~s - ' " , WO 96/09381 . ~ PGTIFT95/Q952~
., ...

electrophoresis, the dried gels were exposed to x-ray film for 2 days.
RNAase protection was accomplished using mouse RNA antisense probes of 291 by and 239 by gene-s rated from linearized plasmids containing the approx-imately 842 by AflII - BamHI and 1.1 kb HindIII - ApaI mouse Tie promoter DNA inserts. The human RNA probe of 568 by was generated from linearized pGEM 3Zf(+) plasmid (Promega, USA) containing an AccI-AlwNI human Tie l0 promoter DNA insert. The template for the other human 266 by RNA probe was generated by PCR
amplification from the AccI-AlwNI plasmid. M13 Forward and Tie 2168 primers (marked in Figure 2) were used for amplification. The probes were labeled 15 using T7 polymerase and ('y-32PJ -UTP. 10 ~g of poly A(+) RNA was incubated with labelled probe at 50°C
overnight. Unhybridized RNA was digested with RNAse A (10 U/ml) and T1 (1 ~Cg/ml) at 37°C, pH 7.5 for 1 hour. The RNAses were inactivated by proteinase K
20 digestion at 37°C for 15 minutes and the samples were analyzed in 8% sequencing gels.
The primer~extension and RNase protection products terminated at positions 101 by and 116 by upstream from ATG codon, in mouse and human Tie 25 promoters, respectively (see asterisks in Figure 2).
? Yeast tRNA or NIH 3T3 RNA did not show any specific bands. Results are shown in Figure 2, wherein the sequences of primers referred to above are underlined.
30 EXAMPhE III
Construction of plasmids Tie promoter/luciferase gene constructs were generated by subcloning the 5' flanking approxi-SU8ST1TUT~ SHEET (RULE 26) .~ .
WO 96/U9381 P~,~~~5~ . ' . _ . ;
... . .. ' mat a ly 788 by genomic AflII-ApaI fragment located upstream of the ApaI restriction site in the first exon to the promoterless basic pGL2 vector (Stratagene) as described by deWet, et al., Mol. Cell. Biol., 7:
725-737 (1987), resulting in plasmid 0.73mTIEpromGL2.
For experiments in transgenic mice, the ap-proximately 788 by AfIII-ApaI promoter fragment (shown in Figure 2) was blunt-end ligated into a blunted, unique HindIII site in the SDK-LacZ Hluescript vector (Stratagene), as described in Logan, et al., Development, 117: 905-916 (1993) resulting in vector 0.73mpromSDK-LacZ. Similarly the 5 kb AlwNI fragment of the human Tie promoter shown in Figure 2 was blunt-end ligated into that same vector, resulting in plasmid S.OhTIEpromSDK-LacZ and deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, as Accession Number 75893 ~XAMPL~ IV
DNA transfection and preparation of cell lysates 15 ug of the 0,73mTIEpromGL2 plasmid described above was transfected into either LE II
25? mouse lung endothelial cells which are described in Schrieber, et al., Proc. Natl. Acad. Sci. (USA), 82:
6138-6142 (1985) or 2~C-2 cells described by weissman, et al., Cell, 32: 599-606 (1983), Transfection was accomplished using the modified calcium phosphate mediated transfection method reported in Sambrook, et a1. (eds.), Molecular Cloning: A Laboratory Manual (1989), The DNAs were SUB~STiTUTE SHEET (RULE 26) WO 96109381 ~ ~ ~ ~ ~ ~ ~ ~PCTJ~T95/08520 , . .,. .. ,. ,.

mixed with 0.25 M CaClZ and an equal volume of 50 mM
N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid-buffered saline. The mixture was incubated for 15 minutes at room temperature and then added dropwise on growing cell monolayers.
The resulting cultures were incubated for 12 hours, after which 5% glycerol in PBS (phosphate buffered saline) was added for 30 seconds and washed off with two changes of PHS. Fresh medium was~then l0 added. After further incubation for 24 hours, the cells were lysed in 0.5 ml lysis buffer (25 mM
Tris-P04, 2 mM dithiothreitol (DTT), 2 mM
1,2-diamino-cyclohexane, N,N,N',N'-tetracetic acid;
l0% glycerol, 1% Triton X-100, pH 7.8). The resulting lysates were centrifuged and the supernatants were collected and stored at 70°C until further assayed. Normalization of luciferase values relative to transfection efficiency was achieved by cotransfection of a CMV-,Q-gal vector described in MacGregor and Casket', Nucl. Acids. Res., 17: 2365 (1989), Assays for ~-galactosidase and luciferase were conducted on transfected cells. For the ~i-galactosidase assay, 30 ml of the cell lysate described above was incubated in 33 ml. of ?o-nitraphenyl-~-D-galactopyranoside (4 mg/ml) dissolved in 100 ml 0.1 M sodium phosphate, pH 7.5 for 30 minutes at 37°C. Optical density was measured at 414 nm.
Luciferase assays were performed using a FlyLight monitoring Kit (102-100, BioTools, Finland) according to the manufacturer's protocol. Briefly, 20 ml of cell lysate was incubated in 100 ml reaction mixture and a Bio-Orbit 1253 luminometer was used to determine light intensity.
SUBSTITUTE SHEET (RULE 26) _.
WO 96/09381 PCT~SIOe52~ ; ' ' ' . ; o ." , "~ ~, The activity of the Tie promoter in cultured cells was measured using Tie promoter-luciferase constructs described above. The Tie promoter-luciferase constructs were transfected into either LEII endothelial cells or into MK-2 epithelial cells. Promoter activity was determined as the ratio of luciferase to ~i-galactosidase activity. Those activities were compared to the promoter activity of the positive control vector, IO RSV-luc (ATCC).
Activity of the 0,73mTIEpromGL2 plasmid (788) relative to CMV-,Q-gal, used as a constitutively expressed cotransfected control promoter, is shown in Figure 3, along with values for the highly expressed RSV-luc promoter. A 460 by mouse Tie promoter fragment was used in reverse orientation as a negative control (reverse). As shown in figure 3, the Tie promoter was highly active in LEII cells but not in the epithelial cells, MK-2. Those results indicate that the isolated Tie promoter is specific for vascular endothelial cells and efficiently promotes the expression of the reporter in those cells in comparison to the control.

Production of transgenic mice The Tie-containing transgene was separated from the vector sequence by digestion with SalI, purified by electrophoresis through an agarose gel, and recovered by absorption on glass beads (Gene Clean II, Bio 101 Inc., La Jolla, CA) according to the manufacturer's instructions. Transgenic mice were produced by the standard microinjection technique reported in Hogan et al., Manipulating the a SU8ST1TUTE SHEET (RULE 26) ~zaa~~s - . . . ; :.
wo 9sro9s8i p~-r~srooszfl ' ' ' ' ' ..
... , mouse embryo (Cold Spring Harbor, 1986), Zygotes for microinjections were obtained from superovulated (BALB/c X DBA/2)F1 hybrid female mice (CD2F1) mated with CD2F1 males. Alternatively, eggs used for injection were from randomly bred superovulated CD1 females. After microinjection, zygotes were transferred at the one or two cell stage into oviducts of pseudopregnant foster mothers (CD2F1 to mice). Tail samples were taken from mouse pups at three weeks of age and DNA Was isolated from the samples by the salt precipitation method of Miller, et al., Nucl. Acids. Res., 16: 1215 (1988), The polymerase chain reaction was used to confirm.the presence of the transgene using the mouse promotez-specific primer, 5'-CTATTGAGAAGGTTTGGAGG3-3'[SEQ ID N0:6), the lacZ
vector primer, 5'-GCTCTAGAACTAGTGGATC-3'[SEQ ID
N0:7]; the human promoter-specific primer, 5'-GAGACAGGGGATGGGAAAAA-3' [SEQ ~ID NO:B]; and the lacZ vector primer, 5'-GAAGATCGCACTCCAGCCAG-3' [SEQ
ID NO: 9] using a reaction mixture comprising 200 ng DNA (Tail)..; lOx buffer (2mM MgCl2), 250 nM Primer 2040, 250 nM Primer 1986, 0.2 mM dNTP Mixture, 0.02 U Dynazyme(Finnzymes, Finland), and 50 ml of distilled water, plus 50 ml mineral oil (M-3516;
Sigma, USA). The PCR Program consisted of a hot start at 96°C, 2 minutes, with cycling as follows:
96°C 1 minutes, 50°C 2 minutes, 72°C 3 minutes, for 34 Cycles, with the last step delayed 10 minutes.
EXAMPLE VI
Analysis of Tie-containing Tissue Whole mouse embryos were obtained and stained for (3-galactosidase activity. Tissue was t SU8S11TUTE SHEET (RULE 26) transferred into 4x paraformaldehyde in PBS (pH 7.4) and incubated at 4°C for 20 minutes with gentle agitation. Tissue was then washed with PBS and incubated in fresh X-Gal reaction mixture [1 mg/ml 4-chloro-5-bromo-3-indolyl-~i-galactoside, 4mM
K4Fe(CN) 6 x 3H20, 2mM MgCl2 in PBS] at 30°C for 1 to 2 days. Then, samples were washed in PBS for 5 hours and transferred to 30% sucrose for storage.
Samples were then embedded in Tissue Tek (Miles, USA) and 15 ~Cm sections were cut on silane-treated slides. Sections were post-fixed in 4% paraformaldehyde for 5 minutes, and washed twice in PBS and once in distilled water. Nuclear fast red was applied as a counterstain.
Results are provided in Figures 5~and 6.
Figures 5A-5D show expression of the mouse Tie promoter in 9.5 (Figures 5A and 5B) and 11.5 (Figures 5C and 5D) day post coitum mouse embryos.
As seen in the figures, activity of the ~i-galactosidase reporter gene is found in the developing heart (h), branchial vessels (ba), paired dorsal aorta (da), vitelline artery (v), umbilical artery (u), and in capillaries (c) of 9.5 day post coitum embryos. Two days later (figures 5C and 5D), a similar pattern is found with the addition of staining in the mesonephros (m) and the veins of the liver(1).
Figures 6A through 6F show Tie promoter activity in 9.5, 11.5, and 13.5 day post coitum embryos. All endothelial cells of the cardiac region are stained, indicating expression under control of the promoter. Staining is observed in lung, but the bronchi are negative. Brain tissue of 15.5 day embryos also shows staining. Figure 6D
shows that the promoter is expressed in veins of the SU85TITUTE SHEET (RULE 26~

liver and Figure 6E shows staining in the developing bone trabeculae. The developing cortex of the kidney shows staining, expression being most prominent in the glomeruli.
To study the promoter activity during development, the 0.73mpromSDK-LacZ and S.OhpromSDK-LacZ DNAs were injected into fertilized mouse oocytes. Six transgenic mice were obtained, transgenic males were mated with wild-type NMRI
females and the offspring (86 for 735 by fragment and 57 for 5.0 kb fragment) were analyzed on days 7.5 - 17.5 of development. Of the F1 offspring, 40%
were positive in LacZ staining, although the embryos showed a variation of the intensity of the reaction color. No staining was seen in 7.5 day post-coital embryos, whereas in 8.5 day post coitum embryos, endothelial cells of the dorsal aorta and forming heart were strongly positive. Certain cells of the head mesenchyme, presumably differentiating angioblasts, showed a faint signal, and the extraembryonic tissues, such as allantois and yolk sac, contained positive vessels.
The complexity of the vascular system increases rapidly in the developing embryo, and in 9.5 day post coitum embryos promoter activity was seen in the above mentioned vessels as well as in the intersomitic arteries. An especially intense staining was seen in the developing ventricles of the heart. In 11.5 day post coital embryos the capillary system is well-developed and therefore the staining associated with large vessels of the embryo and the endocardium was only faintly discerned through the dense network of blue-stained capillaries. The details of vascular system were better visualized in high magnification of tissues SUBSTITUTE SHEET (RULE 26) ;., ~, WO 96/09381 ~ , , ~ PCT/FT95/ON52(~ ~ ' ' ; ;
.., .. ,.. ., of day 11.5 and 15.5 post coital embryos. That staining pattern corresponds to the expression pattern obtained in in situ hybridization. As shown in Figures 4A and 4B, the endocardium of the heart, the veins and the arteries of the head mesenchyme showed LacZ signal. No significant differences were seen in the staining patterns obtained with mouse approximately 788 by and human 5.0 kb promoter fragments.
l0 In order to determine if the promoter ac-tivity of the approximately ~s8 by mouse fragment cor-relates with expression of Tie mRrrA in adult tissues, various tissue types obtained from 8-week old transgenic mice were stained for ,Q-galactosidase activity. As shown in Figures 7A and 7B, intense staining was observed in lung (figure 7A) and bone marrow (designated bm in Figure 7B). Figure 7B also shows staining in capillaries associated with hair follicles (designated by the arrow in Figure 7B).
Slightly less staining was observed in kidney glomeruli (Figure 7C, designated "g") and vessels surrounding the tubuli (Figure 7C, designated by the arrowhead).- Figure 7D shows staining in the endocardium. Neither large hepatic vessels (v in Figure 7E) or sinusoidal capillaries (not shown) stained with LacZ in adult mice. However, small vessels surrounding the veins did stain [Arrows in Figure 7(E)]. ~As shown in Figure 7(F), interstitial capillaries of the brain were stained [Arrowheads in Figure 7(F)]. Similar results were obtained when transgenic mice expressed the 5 kb human Tie promoter.
The present invention has been described in. terms of its preferred, embodiments. Accordingly, the invention should be limited only by the scope of the appended claims.

SU8ST1TUTE SHEET (RULE 26) INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule l3bis) A. T'be indicationc~ade below relate to the microorganism referred t~~ the description Gt5 J

, line .
on page B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet Name of depositary institution American Type Culture Collection (ATCC) Address of depositary institution (including postel code and country) 12301 Parklawn Drive, Rockville, MD 20852, USA

Daze of deposit 20 September 1994 ~sion Number ATCC 75892 C. ADDITIONAL INDICATIONS (leave blank ijnot applicable) This information is continued on an additional sheet In respect of those designations in which a European patent or a patent in Finland or Norway is sought, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or the corresponding information concerning the patent in Finland or Norway or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person re-questing the sample (Rule 28(4) EPC and the corresponding regulations in Finland and Norway).

D. DESIGNATED STATES FOR WINCH
INDICATIONS ARE MADE (ijtlu indications are not jor all daignatcd Starts) E. SEPARATE FURNISHING OF INDICATIONS
(leave blank ijnot applicable) The indications listed below will be submitted to the International Bureau later (specijytJugenerol natareojtheindications eg., ACf:GiftOn Nwrrbn ojDeposit For receiving Office use only For International Bureau use only ~ This sheet was received with the international application ~ This sheet was received by the International Bureau on:
Authorized ofFtcer _ Authorized officer ~,IGuG: /!~ ~e~ ~t.~..
Form PCT/RO1134 (July 1992) INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 136is) A. The indicationc~ade below relate to the microorganism referred t 1~ the description J

e , tin on page B. IDENTIFICATION OF DEPOSIT Further deposiu are identified on an additional sheet Name of depository institution American Type Culture Collection (ATCC) Address of depository institution (including postal code and country) 12301 Parklawn Drive, Rockville, MD 20852, USA

Date of deposit Accession Number 20 September 1994 ATCC 75893 C. ADDTTIONAL IT1DICATIONS (leave blank if not applicable) 'Ibis information is continued on an additional sbeet In respect of those designations in which a European patent or a patent in Finland or Norway is sought, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or the corresponding information concerning the patent in Finland or Norway or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person re-questing the sample (Rule 28(4) EPC and the corresponding regulations in Finland and Norway).

D. DESIGNATED STATES FOR V1~CH
INDICATIONS ARE MADE (i f the indications are not for oll designated Stator) E. SEPARATE FURNISHING OF INDICATIONS
(leave blank if not applicable) The indications listed belowwill be submitted to the International Bureau later (spxifythegaraal notrtreoftheindicotions cg., 'ACCCSfJ0I1 Number of Deposit For receiving Office use only For International Bureau use only ~ This sbeet was received with the international application Q This sheet was received by the International Bureau on:
Authorized offiee~~~ ~ /~~ ~ ~, ~ ~ Authorized officer (~~t/~~-Form PCT/RO/134 (July 1992) Indications relating to deposited microorganisms Continuation to C. ADDITIONAL INDICATIONS
ATCC 75892 and ATCC 75893 When designating Australia, in accordance with regulation 3.25 of the Patents Regulations (Australia Statutory Rules 1991 No. 71), samples of materials deposited in accordance with the Budapest Treaty in relation to this Patent Request are only to be provided before: the patent is granted on the application; or the application has lapsed or been withdrawn or refused; to a person who is: a skilled addressee without an interest in the invention; and nominated by a person who makes a request for the furnishing of those samples.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Helsinki University Licensing Ltd Oy (ii) TITLE OF INVENTION: Promoter for the Receptor Tyrosine Kinase, TIE
(iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Oy Jalo Ant-Wuorinen Ab (B) STREET: Iso Roobertinkatu 4-6 A
(C) CITY: Helsinki (E) COUNTRY: Finland (F) ZIP: 00120 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Karvinen, Leena (C) REFERENCE/DOCKET NUMBER: 28203 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: +358 0 648606 (B) TELEFAX: +358 0 640575 (C) TELEX: 123505 jalo sf (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 882 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

GTTTACTTTTG1~~~P.AAAAAP.AGAGTCACGTGAGCCTCATTTTGTATTTGTGTGTGTGTGT 360 SUBSTITUTE SHEET (RULE 26j (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 935 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY:
linear (ii) MOLECULE
TYPE: DNA (genomic) (xi) SEQUENCE
DESCRIPTION:
SEQ ID N0:2:

(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 131 amino acids (B) TYPE: amino acid SUBSTITUTE S4iEET (RULE 2~

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Gly Cys Val Lys Asp Cys Pro Gly Cys Leu His Gly Gly Val Cys His Asp His Asp Gly Cys Val Cys Pro Pro Gly Phe Thr Gly Thr Arg Cys Glu Gln Ala Cys Arg Glu Gly Arg Phe Gly Gln Ser Cys Gln Glu Gln Cys Pro Gly Thr Ala Gly Cys Arg Gly Leu Thr Phe Cys Leu Pro Asp Pro Tyr Gly Cys Ser Cys Gly Ser Gly Trp Arg Gly Ser Gln Cys Gln Glu Ala Cys Ala Pro Asp His Phe Gly Ala Asp Cys Arg Leu Gln Cys Gln Cys Gln Asn Gly Gly Thr Cys Asp Arg Phe Ser Gly Cys Val Cys Pro Ser Gly Trp His Gly Val His Cys Glu Lys Ser Asp Arg Ile Pro Gln Ile Leu (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:

(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
SUBSTITUTE SHEET (RULE 26) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:

(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:

(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:

(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:

(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:

SUBSTITUTE SHEET (RULE 26)

Claims (16)

What is claimed is:

1. An isolated and purified promoter sequence for a mouse Tie receptor tyrosine kinase, said promoter sequence comprising the nucleic acid sequence of SEQ ID
NO:1.

2. A promoter sequence for a mouse Tie receptor tyrosine kinase, wherein said promoter sequence is a portion of the nucleic acid sequence of SEQ ID NO:1 capable of promoting expression of an endothelial cell receptor tyrosine kinase.

3. The promoter sequence according to claim 1 or 2, wherein said promoter sequence is capable of promoting transcription of a marker gene for use in endothelial cell tagging in a transfected mouse endothelial cells.

4. The promoter sequence according to claim 1, said promoter sequence comprising a Tie promoter insert of plasmid 0.73mTIEpromGL2, wherein said Tie promoter insert is an AfIII - ApaI fragment, and wherein said plasmid has ATCC accession no. 75892.

5. The promoter sequence according to claim 4, wherein said Tie promoter insert is capable of promoting transcription of a protein-encoding DNA for production of proteins and peptides, which act as anticoagulants or vasodilator inhibitors of thrombosis or restenosis, in endothelial cells.

6. A promoter sequence for a human Tie receptor tyrosine kinase, said promoter sequence comprising the nucleic acid sequence of SEQ ID No:2.

7. A promoter sequence for a human Tie receptor tyrosine kinase, wherein said promoter sequence is a portion of the nucleic acid sequence of SEQ ID No:2 capable of promoting expression of an endothelial cell receptor tyrosine kinase.

8. The promoter sequence according to claim 6, said promoter sequence comprising a Tie promoter insert of plasmid 5.OhTIEpromSDK-LacZ, wherein said Tie promoter insert is a 5-kb AIwNI fragment of the human Tie promoter, and wherein said plasmid has ATCC accession no. 75893.

9. The promoter sequence according to claim 8, wherein said Tie promoter insert is capable of promoting transcription of a protein-encoding DNA for production of proteins and peptides, which act as anticoagulants or vasodilator inhibitors of thrombosis or restenosis, in endothelial cells.

10. The promoter sequence according to any one of claims 1-9, wherein said promoter sequence is capable of promoting transcription of an antisense RNA
construct for use in endothelial cells.

11. The promoter sequence according to any one of claims 1-9, wherein said promoter sequence is capable of promoting transcription of a beta-galactosidase reporter gene in endothelial cells of mouse tissue.

12. A vector comprising an isolated and purified promoter sequence according to any one of claims 1-9.

13. Vector 0.73mTIEpromGL2, having ATCC accession number 75892.

14. Vector 5.OhTIEpromSDK-LacZ, having ATCC accession number 75893.

15. A host cell transfected with a promoter sequence according to any one of claims 10-11.

16. A host cell transfected with a vector according to any one of claims 12-14.