Materials and methods for delivery and expression of heterologous DNA in vertebrate cells

Abstract

The subject invention pertains to recombinant entomopox vectors which are useful for the delivery and stable expression of heterologous DNA in vertebrate cells. Specifically exemplified is a recombinant EPV from amsacta moorei (AmEPV). Because of the capacity of the EPV to incorporate foreign or heterologous DNA sequences, the vectors of the subject invention can be used to deliver DNA inserts that are larger than 10 kb in size. Accordingly, one aspect of the present invention concerns use of the recombinant vectors for delivery and expression of biological useful proteins in gene therapy protocols. In addition, the subject invention concerns novel AmEVP polypeptides and the polynucleotide sequences which encode these polypeptides.

1 . A method for delivering a polynucleotide encoding a protein to a vertebrate cell, said method comprising introducing into said vertebrate cell a recombinant entomopox virus vector comprising said polynucleotide operably linked with a promoter sequence, thereby delivering and expressing said polynucleotide encoding said protein in said vertebrate cell.  
     
     
         2 . The method according to  claim 1 , wherein said vertebrate cell is a mammalian cell.  
     
     
         3 . The method according to  claim 2 , wherein said mammalian cell is a human cell.  
     
     
         4 . The method according to  claim 1 , wherein said vector comprises inverted terminal repeat sequences flanking said polynucleotide encoding said protein.  
     
     
         5 . The method according to  claim 4 , wherein said inverted terminal repeat sequences are derived from adeno-associated virus.  
     
     
         6 . The method according to  claim 1 , wherein said promoter sequence is capable of driving expression of said polynucleotide encoding said protein.  
     
     
         7 . The method according to  claim 6 , wherein said promoter sequence is selected from the group consisting of a CMV promoter sequence and herpes TK promoter sequence.  
     
     
         8 . The method according to  claim 1 , wherein said protein encoded by said polynucleotide is selected from the group consisting of interleukins, cytokines, growth factors, interferons, enzymes, and structural proteins.  
     
     
         9 . The method according to  claim 1 , wherein said vector is introduced into said vertebrate cell by infection in a viral particle.  
     
     
         10 . The method according to  claim 1 , wherein said vector is introduced into said vertebrate cell by means selected from the group consisting of transfection, transduction, and injection.  
     
     
         11 . The method according to  claim 1 , wherein said vector is introduced into said vertebrate cell in vivo.  
     
     
         12 . The method according to  claim 1 , wherein said polynucleotide encoding said protein is greater than about 10 kb in size.  
     
     
         13 . The method according to  claim 1 , wherein said polynucleotide also encodes a selectable marker protein.  
     
     
         14 . A vertebrate cell comprising a recombinant entomopox virus vector comprising a polynucleotide encoding a protein operably linked with a heterologous promoter sequence.  
     
     
         15 . The vertebrate cell according to  claim 14 , wherein said cell expresses said protein encoded by said polynucleotide.  
     
     
         16 . A human cell comprising a recombinant entomopox virus vector comprising a polynucleotide encoding a protein operably linked with a non-poxvirus promoter sequence, wherein said non-poxvirus promoter sequence is activated by the cellular RNA polymerase of said human cell.