Novel vectors and genes exhibiting increased expression

Abstract

Disclosed is a liver specific expression vector designed for expression of blood coagulation factor proteins. The expression vector comprises a DNA coding sequence for a blood coagulation factor operably linked to a liver-specific promoter and a liver-specific enhancer, wherein the promoter and enhancer are derived from different genes. In a particular embodiment, the liver-specific promoter is the human thyroid binding globulin promoter and the liver-specific enhancer is the alpha-1 microglobulin/bikunin enhancer. The expression vector may further contain modifications for optimal liver-specific expression.

What is claimed is:  
     
         1 . An expression vector comprising a DNA sequence encoding a blood coagulation factor operably linked to a liver-specific promoter and a liver-specific enhancer, wherein the promoter and enhancer are derived from different genes, and wherein the liver-specific promoter is the human thyroid binding globulin promoter.  
     
     
         2 . The expression vector of  claim 1 , wherein the promoter and enhancer are located upstream of the coding sequence.  
     
     
         3 . The expression vector of  claim 2  wherein the coding sequence is preceded upstream by a leader sequence which has no secondary structure when transcribed as RNA.  
     
     
         4 . The expression vector of  claim 2 , wherein the DNA sequence is expressed as a β-domain deleted human Factor VIII protein.  
     
     
         5 . The expression vector of  claim 1 , wherein the liver-specific enhancer is the alpha-1 microglobulin/bikunin enhancer.  
     
     
         6 . The expression vector of  claim 2  further comprising one or more introns located (a) downstream of the promoter and enhancer and (b) upstream of the coding sequence.  
     
     
         7 . The expression vector of  claim 6 , wherein the coding sequence is preceded upstream by a leader sequence, and the intron is located within the leader sequence.  
     
     
         8 . The expression vector of  claim 6 , wherein the intron comprises one or more consensus splice sites.  
     
     
         9 . The expression vector of  claim 7 , wherein the leader sequence has no secondary structure when transcribed as RNA.  
     
     
         10 . The expression vector of  claim 2  wherein the coding sequence comprises a 3′ untranslated region which is modified to increase processing, export or stability of an mRNA transcribed from the coding sequence.  
     
     
         11 . An expression vector comprising the human thyroid binding globulin promoter and the alpha-1 microglobulin/bikunin enhancer, wherein the human promoter and enhancer are located upstream of a DNA sequence encoding a human Factor VIII protein.  
     
     
         12 . The expression vector of  claim 11  comprising two or more copies of the alpha-1 microglobulin/bikunin enhancer.  
     
     
         13 . The expression vector of  claim 11 , wherein the DNA sequence is also preceded upstream by a leader sequence comprising one or more introns.  
     
     
         14 . The expression vector of  claim 12  wherein the DNA sequence is expressed as a β-domain deleted human Factor VIII protein.  
     
     
         15 . The expression vector of  claim 13 , wherein the intron comprises a consensus 5′ splice donor site, and a consensus 3′ splice acceptor site.  
     
     
         16 . The expression vector of  claim 13 , wherein the intron has no secondary structure when transcribed as RNA.  
     
     
         17 . An expression vector comprising a liver-specific promoter and a liver-specific enhancer, wherein said promoter and enhancer are derived from different genes and are located upstream from a DNA sequence encoding a human Factor VIII protein.  
     
     
         18 . The expression vector of  claim 17 , wherein the DNA sequence is expressed as a β-domain deleted human Factor VIII protein.  
     
     
         19 . The expression vector of  claim 17 , wherein the liver-specific promoter is the human thyroid binding globulin promoter  
     
     
         20 . The expression vector of  claim 17 , wherein the liver-specific enhancer is the alpha-1 microglobulin/bikunin enhancer.  
     
     
         21 . The expression vector of  claim 17 , further comprising one or more introns located (a) downstream of the promoter and enhancer and (b) upstream of the coding sequence.  
     
     
         22 . The expression vector of  claim 21 , wherein the DNA sequence is preceded upstream by a leader sequence, and the intron is located within the leader sequence.  
     
     
         23 . The expression vector of  claim 21 , wherein the intron comprises one or more consensus splice sites.  
     
     
         24 . The expression vector of  claim 22 , wherein the leader sequence has no secondary structure when transcribed as RNA.  
     
     
         25 . The expression vector of  claim 17 , wherein the DNA sequence comprises a 3′ untranslated region which is modified to increase processing, export or stability of the mRNA transcribed from the coding sequence.  
     
     
         26 . The expression vector of  claim 17  comprising two or more copies of the alpha-1 microglobulin/bikunin enhancer.