Screening and Therapy for Lymphatic Disorders Involving the FLT4 Receptor Tyrosine Kinase (VEGFR-3)

Abstract

The present invention provides materials and methods for screening for and treating hereditary lymphedema in human subjects.

1 - 11 . (canceled)  
     
     
         12 . A method of treatment for hereditary lymphedema, comprising: 
 administering to a patient with hereditary lymphedema a therapeutically effective amount of a growth factor product selected from the group consisting of vascular endothelial growth factor C (VEGF-C) protein products, vascular endothelial growth factor D (VEGF-D) protein products, and VEGF-D gene therapy protein products,    wherein said patient with hereditary lymphedema comprises a mutation that alters the encoded amino acid sequence of at least one VEGFR-3 allele of the patient, wherein said mutation reduces ligand-mediated signaling of the VEGFR-3 polypeptide encoded by the allele, when compared to VEGFR-3 encoded by a wild-type human VEGFR-allele; and    wherein said therapeutically effective amount of said growth factor product is administered locally at a site of edema in the patient.    
     
     
         13 . A therapeutic or prophylactic method of treating lymphedema, comprising the steps of: 
 providing isolated lymphatic endothelial cells or lymphatic endothial progenitor cells;    transforming or transfecting the cells ex vivo with a polynucleotide comprising a nucleotide sequence that encodes a wild type VEGFR-3;    and administering the transformed or transfected cells to the mammalian subject.    
     
     
         14 - 21 . (canceled)  
     
     
         22 . A purified polynucleotide comprising a nucleotide sequence encoding a human VEGFR-3 protein variant, wherein said polynucleotide is capable of hybridizing to the complement of SEQ ID NO: 1 under the following hybridization conditions: 
 hybridization at 42° C. in 50% formamide, 5×SSC, 20 mM Na·PO 4 , pH 6.8; and    washing in 0.2×SSC at 55° C.;    and wherein the encoded VEGFR-3 protein variant has an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO:2 at one or more positions selected from the group consisting of amino acids 843 to 943 of SEQ ID NO: 2 and amino acids 1009 to 1165 SEQ ID NO: 2.    
     
     
         23 - 29 . (canceled)  
     
     
         30 . A method for identifying a modulator of intracellular VEGFR-3 signaling, comprising the steps of: 
 a) contacting a cell expressing at least one mutant mammalian VEGFR-3 polypeptide in the presence and in the absence of a putative modulator compound;    b) detecting VEGFR-3 signaling in the cell; and    c) identifying a putative modulator compound in view of decreased or increased signaling in the presence of the putative modulator, as compared to signaling in the absence of the putative modulator.    
     
     
         31 - 36 . (canceled)  
     
     
         42 . The method of  claim 12  or  37 , wherein the wildtype VEGFR-3 allele comprises the VEGFR-3 coding sequence set forth in SEQ ID NO: 1.  
     
     
         43 . The method of  claim 12  or  37 , wherein said administering of said therapeutically effective amount of said growth factor product induces VEGF-3 signaling in the lymphatic endothelia of the patient.  
     
     
         44 . The method of  claim 12  or  37 , wherein said administering of said therapeutically effective amount of said growth factor product reduces edema in a limb of said patient.  
     
     
         45 . The method of  claim 12  or  37 , wherein said administering of said therapeutically effective amount of said growth factor product reduces accumulation of lymph fluids in said patient.  
     
     
         46 . The method of  claim 45 , wherein the administering step comprises administering a composition that comprises the growth factor product and a pharmaceutically acceptable carrier.  
     
     
         47 . The method of  claim 46 , wherein said growth factor product comprises a VEGF-D protein product comprising a member selected from the group consisting of: 
 (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 6;    (b) a polypeptide that comprises an amino acid sequence that comprises a continuous portion of SEQ ID NO: 6 sufficient to permit the polypeptide to bind wildtype human VEGFR-3 and stimulate VEGFR-3 phosphorylation in cells that express wildtype human VEGFR-3;    (c) a polypeptide of (a) or (b), with the proviso that the polypeptide has up to 25 amino acids added, deleted, or substituted with another amino acid, compared to the amino acid sequence of the polypeptide of (a) or (b), and wherein the polypeptide retains the ability to bind and stimulate phosphorylation of wildtype human VEGFR-3.    
     
     
         48 . The method of  claim 47 , wherein the VEGF-D protein product comprises amino acids 93-201 of SEQ ID NO: 6.  
     
     
         49 . The method of  claim 47 , wherein said growth factor product is administered via intravenous injection.  
     
     
         50 . The method of  claim 47 , wherein said growth factor product is administered via intramuscular injection.