System for monitoring the expression of transgenes

Abstract

A novel strategy for monitoring the expression of a transgene in a patient is disclosed. A marker peptide is genetically fused to either the N-terminus or C-terminus of the product of a transgene through a linker peptide which bears the recognition sequence of a host cell protease. Expression of the transgene results in release of the marker peptide into extracellular body fluid of the patient in proportion to the amount of transgene product. The level of the released marker peptide serves as an indicator of the level of transgene expression. The system is particularly useful for monitoring the expression of therapeutic transgenes and production of the therapeutic gene product.

1 - 56  (Cancelled).  
     
     
         57 . A nucleic acid construct comprising a transgene and a sequence encoding a marker polypeptide, wherein said marker polypeptide is releasable from cells into extracellular body fluid when expressed in a mammal, and wherein either: 
 (a) a first promoter regulates expression of said transgene, and a second promoter regulates expression of the sequence encoding said marker polypeptide, or    (b) a promoter regulates expression of said transgene and the sequence encoding said marker polypeptide, said transgene and the sequence encoding said marker polypeptide being heterologous.    
     
     
         58 . The nucleic acid construct of  claim 57 , wherein a first promoter regulates expression of said transgene, and wherein a second promoter regulates expression of the sequence encoding said marker polypeptide.  
     
     
         59 . The nucleic acid construct of  claim 57 , wherein said nucleic acid comprises, between said transgene and the sequence encoding said marker polypeptide, an internal ribosomal entry site.  
     
     
         60 . The nucleic acid construct of  claim 57 , wherein said marker polypeptide is derived from a peptide hormone or peptide hormone precursor by proteolytic cleavage or mutation of one or more amino acid residues.  
     
     
         61 . The nucleic acid construct of  claim 57 , wherein said marker polypeptide is insulin C-peptide.  
     
     
         62 . The nucleic acid construct of  claim 57 , wherein said marker polypeptide is derived from parathyroid hormone or a fragment of parathyroid hormone by proteolytic cleavage or mutation of one or more amino acid residues.  
     
     
         63 . The nucleic acid construct of  claim 57 , wherein said marker polypeptide is beta HCG or a fragment thereof.  
     
     
         64 . The nucleic acid construct of  claim 57 , wherein said transgene encodes a fusogenic polypeptide.  
     
     
         65 . The nucleic acid construct of  claim 64 , wherein said fusogenic polypeptide is a viral fusion protein.  
     
     
         66 . The nucleic acid construct of  claim 64 , wherein said fusogenic polypeptide is a gibbon ape leukemia virus envelope glycoprotein.  
     
     
         67 . The nucleic acid construct of  claim 64 , wherein said fusogenic polypeptide is a measles virus H glycoprotein.  
     
     
         68 . The nucleic acid construct of  claim 57 , wherein said nucleic acid construct comprises, between said transgene and the sequence encoding said marker polypeptide, a sequence encoding a protease-cleavable linker.  
     
     
         69 . The nucleic acid construct of  claim 68 , wherein said protease-cleavable linker comprises an auto-cleaving sequence.  
     
     
         70 . The nucleic acid construct of  claim 68 , wherein the sequence encoding said protease-cleavable linker is fused in-frame to the 5′ end of said transgene.  
     
     
         71 . The nucleic acid construct of  claim 68 , wherein the sequence encoding said protease-cleavable linker is fused in-frame to the 3′ end of said transgene.  
     
     
         72 . The nucleic acid construct of  claim 68 , wherein said protease-cleavable linker is identical to a linker present in a cell surface protein or secretory protein.  
     
     
         73 . The nucleic acid construct of  claim 68 , wherein said protease-cleavable linker is cleaved by furin.  
     
     
         74 . The nucleic acid construct of  claim 68 , wherein said protease-cleavable linker is identical to a linker present in a cytoplasmic protein.  
     
     
         75 . A host cell comprising the nucleic acid construct of  claim 57 .  
     
     
         76 . A kit comprising: 
 (a) the nucleic acid construct of  claim 57  or a cell transfected with said nucleic acid construct, and    (b) a reagent for monitoring the release of the marker polypeptide.