Regulatory sequences capable of conferring expression of a heterologous DNA sequence in endothelial cells and uses thereof

Abstract

Described are recombinant DNA molecules comprising the regulatory sequence(s) of an intron of the Endothelial Growth Factor (VEGF) receptor- 2 gene (Flk- 1 ) or of a gene homologous to the Flk- 1 gene, being capable of conferring expression of a heterologous DNA sequence in endothelial cells, preferably in vivo. Vectors comprising said DNA molecules as well as host cells containing the same are provided. Also provided are pharmaceutical and diagnostic compositions comprising such recombinant DNA molecules and vectors. Furthermore, cells and transgenic non-human animals, comprising the aforementioned recombinant DNA molecules or vectors stably integrated into their genome and their use for the identification of substances capable of suppressing or activating transcription of a gene in endothelial cells are described. Described is further the use of the before described recombinant DNA molecules and vectors for the preparation of pharmaceutical compositions for treating, preventing, and/or delaying a vascular or tumorous disease in a subject. Furthermore, uses of the recombinant DNA molecules and vectors of the invention for the preparation of pharmaceutical compositions for inducing a vascular or tumorous disease in a non-human animal are provided.

1 . A recombinant DNA molecule comprising: 
 (a) at least one first regulatory sequence of an intron which confers expression in endothelial cells in vivo, wherein said first regulatory sequence is selected from the group consisting of 
 (i) a DNA sequence comprising a nucleotide sequence as given in SEQ ID NO: 1;  
 (ii) a DNA sequence comprising a nucleotide sequence of SEQ ID NO:1 from nucleotide 8260 to nucleotide 10560 from nucleotide 8336 to nucleotide 10608 and/or from nucleotide 10094 to nucleotide 10608; and  
 (iii) a DNA sequences comprising a fragment of a nucleotide sequence of (i) or (ii); and  
   (b) operatively linked thereto a heterologous DNA sequence.    
     
     
         2 . The recombinant DNA molecule of  claim 1 , wherein said first regulatory sequence comprises a GATA-binding site, an AP-1 binding site, an SP1 binding site, an NFκB binding site, a STAT binding site, a Scl/tal-1 binding site, an Ets-1 binding site, a PEA3 consensus sequence or any combination(s) thereof.  
     
     
         3 . The recombinant DNA molecule of  claim 1  or  2 , wherein said first regulatory sequence is selected from the group consisting of 
 (a) a DNA sequence comprising a nucleotide sequence as given in SEQ ID NO:1;    (b) a DNA sequence comprising a nucleotide sequence of SEQ ID NO:1 from nucleotide 8260 to nucleotide 10560, from nucleotide 8336 to nucleotide 10608 and/or from nucleotide 10094 to nucleotide 10608; and    (c) a DNA sequence comprising a fragment of a nucleotide sequence of any one of (a) or (b) that confers expression in endothelial cells.    
     
     
         4 . The recombinant DNA molecule of any one of  claims 1  to  2  wherein said heterologous DNA sequence is operatively linked to further regulatory sequences.  
     
     
         5 . The recombinant DNA molecule of  claim 4 , wherein said further regulatory sequence is a promoter.  
     
     
         6 . The recombinant DNA molecule of  claim 4 , wherein said further regulatory sequence is a 3′-untranslated region.  
     
     
         7 . The recombinant DNA molecule of  claim 5 , wherein said promoter is a promoter of hypoxia inducible genes, genes encoding growth factors or its receptors or glycolytic enzymes.  
     
     
         8 . The recombinant DNA molecule of  claim 7 , wherein said growth factor is VEGF, PDGF or Fibroblast growth factor.  
     
     
         9 . The recombinant DNA molecule of  claim 5 , wherein said promoter comprises a DNA sequence selected from the group consisting of 
 (a) a DNA sequence comprising the nucleotide sequence as given in SEQ ID NO:1 from nucleotide 6036 to nucleotide 6959;    (b) a DNA sequence comprising the nucleotide sequence of the human Flk-1/KDR promoter; and    (c) a DNA sequence comprising a fragment of a nucleotide sequence of any one of (a) or (b).    
     
     
         10 . The recombinant DNA molecule of any of  claims 1  to  2 , wherein at least one of said DNA sequences is of human or murine origin.  
     
     
         11 . The recombinant DNA molecule of any one of  claims 1  to  2 , wherein said heterologous DNA sequence being operatively linked to said regulatory sequences is located 5′ to said first regulatory sequence.  
     
     
         12 . The recombinant DNA molecule of  claim 1 , wherein said heterologous DNA sequence encodes a peptide, protein, antisense RNA, sense RNA and/or ribozyme.  
     
     
         13 . The recombinant DNA molecule of  claim 42 , wherein said protein is selected from the group consisting of Vascular Endothelial Growth Factor (VEGF), Hypoxia Inducible Factors (HIF), HIF-Related Factor (HRF), tissue plasminogen activator, p21 cell cycle inhibitor, nitric oxide synthase, interferon-γ, atrial natriuretic polypeptide monocyte chemotactic proteins luciferase green fluorescent protein and lacZ.  
     
     
         14 . The recombinant DNA molecule of  claim 42 , wherein said protein is a scorable marker, preferably luciferase, green fluorescent protein or lacZ.  
     
     
         15 . The recombinant DNA molecule of  claim 12 , wherein said antisense RNA or said ribozyme are directed against a gene involved in vasculogenesis and/or angiogenesis and/or tumors of endothelial origin.  
     
     
         16 . A nucleic acid molecule of at least 15 nucleotides in length hybridizing specifically with the first regulatory sequence of a recombinant DNA molecule of  claim 1 .  
     
     
         17 . A vector comprising a recombinant DNA molecule of any one of  claims 1  to  2 .  
     
     
         18 . The vector of  claim 17 , which is an expression vector and/or a targeting vector.  
     
     
         19 . The vector of  claim 17 , further comprising a gene capable of expressing HIF-2α.  
     
     
         20 . An isolated cell transformed with a DNA molecule of any one of  claims 1  to  2 .  
     
     
         21 . The isolated cell of  claim 20 , which is a prokaryotic or eukaryotic cell.  
     
     
         22 . The isolated cell of  claim 20 , which is an endothelial cell.  
     
     
         23 . The isolated cell of  claim 20  further comprising a recombinant DNA molecule or vector contain a gene capable of expressing HIF-2α.  
     
     
         24 . A pharmaceutical composition comprising a recombinant DNA molecule of  claim 1 , the vector of  claim 17  and/or the nucleic acid molecule of  claim 16  and optionally a pharmaceutically acceptable carrier.  
     
     
         25 . A diagnostic composition comprising a recombinant DNA molecule of  claim 1 , the vector of  claim 17 , the cell of  claim 20  and/or the nucleic acid molecule of  claim 16 , and optionally suitable means for detection.  
     
     
         26 . A method for the production of a transgenic non-human animal, comprising introduction of a recombinant DNA molecule of  claim 1  or a vector of  claim 17  into a germ cell, an embryonic cell or an egg cell or a cell derived therefrom.  
     
     
         27 . A transgenic non-human animal comprising stably integrated into its genome a recombinant DNA molecule of  claim 1  and/or the vector of  claim 17  or obtained according to the method of  claim 26 .  
     
     
         28 . The method of  claim 26  wherein said animal is a mouse.  
     
     
         29 . A method for the identification of a chemical and/or biological substance capable of suppressing the transcription of a gene in endothelial cells comprising: 
 (a) contacting a cell of  claim 20  or the transgenic non-human animal of  claim 27  either of which is capable of expressing the heterologous DNA sequence with a plurality of compounds; and    (b) determining those compounds which suppress the expression of said heterologous DNA sequence.    
     
     
         30 . A method for the identification of a chemical and/or biological substance capable of activating and/or enhancing the transcription of a gene in endothelial cells comprising: 
 (a) contacting a cell of  claim 20  or the transgenic non-human animal of  claim 27  either of which is capable of expressing the heterologous DNA sequence with a plurality of compounds; and    (b) determining those compounds which are capable of activating and/or enhancing the expression of said heterologous DNA sequence.    
     
     
         31 . Use of a recombinant DNA molecule of  claim 15 , the vector of  claim 17 , the cell of  claim 20 , the pharmaceutical composition of  claim 24 , the diagnostic composition of  claim 25  and/or the transgenic non-human animal of  claim 27  for the identification of a chemical and/or biological substance capable of suppressing or activating and/or enhancing the transcription, expression and/or activity of genes and/or its expression products in endothelial cells.  
     
     
         32 . The method of  claim 29 , wherein the chemical and/or biological substance is selected from the group consisting of peptides, proteins, nucleic acids, antibodies, small organic compounds, hormones, neurotransmitters, peptidomimics and PNAs.  
     
     
         33 . A method for the production of a pharmaceutical composition comprising the steps of the method of  claim 29  and (c) formulating the compound identified in step (b) in a pharmaceutically acceptable form.  
     
     
         34 . A method of inhibiting a vascular disease in a subject, comprising contacting an artery of said mammal with the vector of,  claim 17  wherein said heterologous DNA sequence encodes a protein that reduces or prevents the development of the vascular disease.  
     
     
         35 . The method of  claim 34 , wherein said protein reduces proliferation of smooth muscle cells.  
     
     
         36 . Use of a recombinant DNA molecule of  claim 1 , the vector of  claim 17 , the nucleic acid molecule of  claim 16  and/or a substance identified by the method of  claim 29  for the preparation of a composition for directing or preventing expression of genes specifically in endothelial cells.  
     
     
         37 . Use of a recombinant DNA molecule of  claim 1 , the vector of  claim 17 , the nucleic acid molecule of  claim 16  and/or a substance identified by the method of  claim 29  for the preparation of a pharmaceutical composition for treating, preventing and/or delaying a vascular disease and/or a tumorous disease in a subject.  
     
     
         38 . Use of a recombinant DNA molecule of  claim 1 , the vector of  claim 17  and/or the nucleic acid molecule of  claim 16  for the preparation of a pharmaceutical composition for inducing a vascular disease in a non-human animal or in the transgenic non-human animal of  claim 27 .  
     
     
         39 . The method of  claim 34  or the use of  claim 36 , wherein the vascular disease is atherosclerosis and/or a neuronal disorder.  
     
     
         40 . Use of a regulatory sequence as defined in  claim 1  for enhancing and/or directing gene expression in endothelial cells.  
     
     
         41 . The transgenic non-human animal of  claim 27 , where said animal is a mouse.  
     
     
         42 . The recombinant DNA molecule of any one of  claims 1  to  2 , wherein said heterologous DNA sequence encodes a peptide, protein, sense RNA, or ribozyme.  
     
     
         43 . The recombinant DNA molecule of  claim 1 , wherein the first regulatory sequence confers endothelium-specific expression in vivo of the heterologous DNA sequence.  
     
     
         44 . An isolated cell transformed with the vector of  claim 17 .  
     
     
         45 . A recombinant DNA molecule comprising at least one first regulatory sequence which confers expression in endothelial cells in vivo, wherein the first regulatory sequence is a DNA sequence comprising a nucleotide sequence which hybridizes with a nucleotide sequence of  claim 1 , element (i) or (ii) under stringent conditions, and wherein the recombinant DNA molecule further comprises a heterologous DNA sequence operatively linked to the first regulatory sequence.