Plasmids suitable for IL-2 expression

Abstract

The present invention relates to plasmids suitable for IL-2 expression, particularly, human IL-2 expression, and related methods.

What is claimed is:  
     
         1 . A plasmid suitable for IL-2 eukaryotic expression, consisting essentially of: 
 an expression facilitating sequence derived from the immediate-early promoter region of CMV;    an expression facilitating sequence derived from the transcriptional termination/polyadenylation signal sequence of the BGH gene;    a sequence coding for the eukaryotic expression of an IL-2, characterized as possessing a bioactivity of the complete IL-2, operably linked to both of said expression facilitating sequences,    wherein said sequence coding for the expression of an IL-2 is a sequence encoding a mature IL-2 and a non-IL-2 leader peptide that augments eukaryotic expression compared to a wild-type IL-2 leader peptide; and, optionally: 
 a non-mammalian origin of replication; and a sequence operably encoding a selectable marker.  
   
     
     
         2 . The mature IL-2 of  claim 1  that is homologous to wild-type human IL-2.  
     
     
         3 . The plasmid of  claim 1  wherein said non-IL-2 leader peptide constitutes the human IL-2 leader peptide except for the addition of four amino acids.  
     
     
         4 . The plasmid of  claim 1  wherein said non-IL-2 leader peptide constitutes the human IL-2 leader peptide except for the replacement at the 5′ end of Met-Ala-Leu-Trp-Ile-Asp for Met-Tyr.  
     
     
         5 . The plasmid of  claim 1  wherein said expression facilitating sequence derived from the immediate-early promoter region of CMV is a promoter sequence and an intron sequence.  
     
     
         6 . The plasmid of  claim 1  wherein said expression facilitating sequence derived from the transcriptional termination/polyadenylation signal sequence of the BGH gene is a transcriptional termination and a polyadenylation signal sequence.  
     
     
         7 . The plasmid of  claim 1  possessing the functional characteristics of the plasmid encoded by the nucleotide sequence of SEQ ID NO:1.  
     
     
         8 . The plasmid of  claim 1  having the nucleotide sequence of SEQ ID NO:1.  
     
     
         9 . The plasmid of  claim 1  capable of directing production and secretion of human IL-2 in a human tumor cell.  
     
     
         10 . A host cell transformed with the plasmid of  claim 1 .  
     
     
         11 . A method for producing the plasmid of  claim 1 , comprising the steps of: 
 growing bacterial cells containing said plasmid; and    recovering said plasmid from said bacterial cells.    
     
     
         12 . A cassette system adapted for use in the direct intralesional administration of recombinant genes into established tumor cells in vivo, as they grow in situ, to produce and secrete IL-2, consisting essentially of: 
 a transcription unit, encoding IL-2 in a replicon, containing a sequence encoding a non-IL-2 leader peptide constituting the human IL-2 leader peptide except for the replacement at the 5′ end of Met-Ala-Leu-Trp-Ile-Asp for Met-Tyr, which sequence encoding said non-IL-2 leader peptide is operably linked to sequence encoding said IL-2.    
     
     
         13 . A vector comprising a DNA sequence operably encoding a protein of interest and a non-IL-2 leader peptide, which non-IL-2 leader peptide constitutes the human IL-2 leader peptide except for the replacement at the 5′ end of Met-Ala-Leu-Trp-Ile-Asp for Met-Tyr, wherein the non-IL-2 leader peptide is capable of augmenting the secretion of said protein in a mammalian cell.  
     
     
         14 . A pharmaceutical composition comprising the plasmid of  claim 1  in combination with a pharmaceutically acceptable vehicle.  
     
     
         15 . The pharmaceutical composition of  claim 14 , wherein the plasmid is complexed to a cationic lipid mixture.  
     
     
         16 . The pharmaceutical composition of  claim 15 , wherein the cationic lipid mixture is DMRIE-DOPE.  
     
     
         17 . The pharmaceutical composition of  claim 16 , wherein the DMRIE-DOPE has a molar ratio of about 1:1.  
     
     
         18 . The pharmaceutical composition of  claim 15 , wherein the plasmid to lipid ratio is about 5:1.  
     
     
         19 . A eukaryotic expression vector for the expression of a DNA sequence in a human tissue, consisting essentially of: 
 a CMV immediate early sequence that contains a CMV promoter and a CMV intron;    a sequence whose 5′ end is attached to the 3′ end of the CMV sequence, said sequence encoding a non-IL-2 leader peptide, said peptide constituting the human IL-2 leader peptide except for the replacement at the 5′ end of Met-Ala-Leu-Trp-Ile-Asp for Met-Tyr;    a cassette whose 5′ end is attached to the 3′ end of the sequence encoding a non-IL-2 leader peptide, said cassette containing a DNA sequence that is to be expressed; and    a BGH gene sequence whose 5′ end is attached the 3′ end of the cassette, said BGH sequence containing a transcriptional termination and a polyadenylation signal; and    a selectable marker;    wherein said vector is capable of replicating in prokaryotes.    
     
     
         20 . In a method of gene therapy, the improvement comprising administering the plasmid of  claim 1  directly into a solid tumor resulting in the local secretion of said IL-2.