Compositions and methods for the expression of factor VIII polypeptides and uses therefor

Abstract

Compositions and methods are provided for the in vivo gene delivery of nucleic acid sequences encoding the factor VIII protein to the liver endothelial sinusoidal cells (LSECs). Compositions and methods are also provided for the ex vivo gene transfer of nucleic acid sequences encoding the factor VIII protein to cultured LSECs and the implantation of the transformed LSECs in vivo. These methods and compositions increase the level of factor VIII in the blood stream and find use in the gene therapy treatment of hemophilia A.

What is claimed is:  
     
         1 . A method for the expression of a polypeptide comprising: 
 a) stably introducing into an isolated LSEC a DNA construct comprising a nucleotide sequence encoding factor VIII or a functional variant thereof; and,    b) culturing said LSEC under conditions such that the factor VIII polypeptide or a functional derivative thereof is expressed and secreted from said LSEC.    
     
     
         2 . The method of  claim 1 , wherein the culturing of said LSECs occurs in vitro.  
     
     
         3 . The method of  claim 1 , wherein the culturing of said LSECs occurs in vivo.  
     
     
         4 . The method of  claim 1 , wherein said DNA construct further comprises a promoter operably linked to the nucleotide sequence encoding factor VIII or a functional variant thereof.  
     
     
         5 . The method of  claim 4 , wherein said promoter is an endothelial-preferred promoter.  
     
     
         6 . The method of  claim 4 , wherein said promoter is a constitutive promoter.  
     
     
         7 . The method of  claim 1 , wherein said DNA construct is contained in a gene delivery vehicle.  
     
     
         8 . The method of  claim 7 , wherein said gene delivery vehicle is a non-viral vector.  
     
     
         9 . The method of  claim 7 , wherein said gene delivery vehicle is a viral vector.  
     
     
         10 . The method of  claim 9 , wherein said viral vector is selected from the group consisting of a retrovirus, an adeno-associated virus, and an adenovirus.  
     
     
         11 . The method of  claim 1 , wherein said DNA construct is introduced into said LSEC by liposome-mediated transfection, polybrene-mediated transfection, DEAE dextran-mediated transfection, electroporation, calcium phosphate precipitation, micorinjection, or velocity driven microprojectiles.  
     
     
         12 . A method of increasing the level of a factor VIII polypeptide or functional variant thereof in the blood stream of a subject comprising: 
 a) stably introducing into an isolated LSEC a DNA construct comprising a nucleic acid sequence encoding the factor VIII polypeptide or a functional variant thereof, and,    b) implanting said LSECs into a subject in need thereof, wherein implantation of said LSECs results in an increased level of factor VIII polypeptide in the blood of said subject.    
     
     
         13 . The method of  claim 12 , wherein said DNA construct is administered by portal vein injection.  
     
     
         14 . The method of  claim 12 , wherein said DNA construct is contained in a gene delivery vehicle.  
     
     
         15 . The method of  claim 14 , wherein said gene delivery vehicle is a virus.  
     
     
         16 . The method of  claim 15 , wherein said gene delivery vehicle is selected from the group consisting of a retrovirus, an adeno-associated virus, and an adenovirus.  
     
     
         17 . The method of  claim 14 , wherein said gene delivery vehicle is a non-viral vector.  
     
     
         18 . The method of  claim 12 , wherein said DNA construct further comprises a promoter operably linked to the nucleotide sequence encoding the actor VIII polypeptide or functional variant thereof.  
     
     
         19 . The method of  claim 12 , wherein said subject has a factor VIII deficiency.  
     
     
         20 . The method of  claim 19 , wherein said factor VIII deficiency is hemophilia A.  
     
     
         21 . An isolated LSEC having stably incorporated a DNA construct comprising a nucleotide sequence encoding a factor VIII polypeptide or a functional variant thereof, operably linked to a promoter active in said LSEC.  
     
     
         22 . The transformed LSEC of  claim 21 , wherein said promoter is endothelial-specific.  
     
     
         23 . The transformed LSEC of  claim 21 , wherein said promoter is constitutive.  
     
     
         24 . The transformed LSEC of  claim 21 , wherein said cell is from a mammal.  
     
     
         25 . The transformed LSEC of  claim 21 , wherein said cell is from a human.