Hybrid hepatocyte growth factor gene having high expression efficiency of two heterotypes of hepatocyte growth factor

Abstract

The present invention relates to a hybrid Hepatocyte Growth Factor (HGF) gene which is prepared by inserting an inherent or foreign intron between exons 4 and 5 in HGF cDNA, which has a base sequence of SEQ ID NO: 2. The gene has high expression efficiency and simultaneously expresses two heterotypes of HGF and dHGF (deleted variant HGF). Further the gene may be used for treating or preventing ischemic or liver diseases.

What is claimed is: 
     
       1. A method for co-expressing two heterotypes of Hepatocyte Growth Factor (HGF) in vivo in a mammalian subject with ischemic disease in order to treat the disease, comprising: 
 transforming or transfecting to a DNA construct into a cell of a mammalian subject with ischemic disease by administering a DNA construct to the subject, the DNA construct comprising: 
 (a) a promoter, 
 (b) a first cDNA which has the same sequence as exons 1-4 of the human HGF gene wherein said exons 1-4 are arranged in sequential order without an intron therebetween, or degenerates thereof which do not alter the amino acid sequence encoded by said first cDNA, 
 (c) a polynucleotide that has the same sequence as intron 4 of the HGF gene or a functional fragment thereof, and 
 (d) a second cDNA which has the same sequence as exons 5-18 of the human HGF gene wherein said exons 5-18 are arranged in sequential order without an intron therebetween, or degenerates thereof which do not alter the amino acid sequence encoded by said second cDNA; 
 wherein (c) is located between (b) and (d); and the HGF construct simultaneously encodes two heterotypes of human HGF, and 
 whereby two heterotypes of human HGF are co-expressed within the mammalian subject, thereby treating the ischemic disease. 
 
     
     
       2. The method of  claim 1 , wherein said intron has the same sequence as a fragment of intron 4 of the HGF gene. 
     
     
       3. The method of  claim 2 , wherein the construct comprises a nucleotide sequence not less than 90% identical to SEQ ID NO: 19. 
     
     
       4. The method of  claim 3 , wherein the construct comprises a nucleotide sequence not less than 95% identical to SEQ ID NO: 19. 
     
     
       5. The method of  claim 4 , wherein the construct comprises the sequence of SEQ ID NO: 19. 
     
     
       6. The method of  claim 2 , wherein the construct comprises a nucleotide sequence not less than 90% identical to SEQ ID NO: 20. 
     
     
       7. The method of  claim 6 , wherein the construct comprises a nucleotide sequence not less than 95% identical to SEQ ID NO: 20. 
     
     
       8. The method of  claim 7 , wherein the construct comprises the sequence of SEQ ID NO: 20. 
     
     
       9. The method of  claim 2 , wherein the construct comprises a nucleotide sequence not less than 90% identical to SEQ ID NO: 21. 
     
     
       10. The method of  claim 9 , wherein the construct comprises a nucleotide sequence not less than 95% identical to SEQ ID NO: 21. 
     
     
       11. The method of  claim 10 , wherein the construct comprises the sequence of SEQ ID NO: 21. 
     
     
       12. The method of  claim 1 , wherein the one intron has the same sequence as the full intron 4 of the HGF gene. 
     
     
       13. The method of  claim 1 , wherein the construct comprises a nucleotide sequence not less than 90% identical to SEQ ID NO: 2. 
     
     
       14. The method of  claim 13 , wherein the construct comprises a nucleotide sequence not less than 95% identical to SEQ ID NO: 2. 
     
     
       15. The method of  claim 14 , wherein the construct comprises the sequence of SEQ ID NO: 2. 
     
     
       16. The method of  claim 1 , wherein the construct further comprises a terminator sequence, a self-replication sequence, or a secretory signal. 
     
     
       17. The method of  claim 1 , wherein the expression efficiency of the construct is higher than the expression efficiency of HGF cDNA or deleted variant HGF (dHGF) cDNA. 
     
     
       18. The method of  claim 1 , wherein the expression level of the construct is about 20- to 100-fold higher than the expression level of the HGF cDNA or dHGF cDNA. 
     
     
       19. The method of  claim 1 , wherein the cell is a mammalian cell, a bacterial cell or a yeast cell. 
     
     
       20. The method of  claim 19 , wherein the cell is a mammalian cell. 
     
     
       21. The method of  claim 20 , wherein the transformation of said mammalian cell is in vivo.