Targeted introduction of DNA into primary or secondary cells and their use for gene therapy and protein production

Abstract

The present invention relates to a method of gene or DNA targeting in cells of vertebrate, particularly mammalian, origin. That is, it relates to a method of introducing DNA into primary or secondary cells of vertebrate origin through homologous recombination or targeting of the DNA, which is introduced into genomic DNA of the primary or secondary cells at a preselected site. The present invention further relates to primary or secondary cells, referred to as homologously recombinant (HR) primary or secondary cells, produced by the present method and to uses of the homologously recombinant primary or secondary cells. The present invention also relates to a method of turning on a gene present in primary cells, secondary cells or immortalized cells of vertebrate origin, which is normally not expressed in the cells or is not expressed at significant levels in the cells.

1 . A method of introducing exogenous DNA into a preselected site of the genome of a primary or a secondary cell of vertebrate origin, comprising the steps of: 
 a) transfecting the primary or the secondary cell with a DNA construct comprising exogenous DNA which includes DNA sequences homologous to genomic DNA sequences of the primary or secondary cell, thereby producing transfected primary or secondary cells and    b) maintaining transfected primary or secondary cells under conditions appropriate for homologous recombination between DNA sequences in the DNA construct and genomic DNA to occur;    thereby producing homologously recombinant primary or secondary cells.    
     
     
         2 . The method of  claim 1  wherein the primary or secondary cell of vertebrate origin is selected from the group consisting of: fibroblasts, keratinocytes, epithelial cells, endothelial cells, glial cells, neural cells, formed elements of the blood, muscle cells, hepatocytes and precursors thereof.  
     
     
         3 . The method of  claim 2  wherein the primary or secondary cell is of mammalian origin.  
     
     
         4 . The method of  claim 3  wherein the primary or secondary cell is selected from the group consisting of: primary human cells, secondary human cells, primary mouse cells, secondary mouse cells, primary rabbit cells and secondary rabbit cells.  
     
     
         5 . The method of  claim 4  wherein the exogenous DNA encodes a therapeutic product selected from the group consisting of: enzymes, cytokines, hormones, antigens, antibodies, clotting factors, regulatory proteins, ribozymes, transcription proteins, receptors, anti-sense nucleic acid sequences and novel proteins.  
     
     
         6 . The method of  claim 4  wherein the exogenous DNA is itself a therapeutic product selected from the group consisting of DNA sequences sufficient for sequestration of a protein or nucleic acid in the cell, DNA sequences which bind to a cellular regulatory protein, DNA sequences which alter secondary or tertiary chromosomal structure and DNA sequences which are transcriptional regulatory elements.  
     
     
         7 . The method of  claim 1  wherein the DNA construct of (a) additionally includes DNA encoding a selectable marker.  
     
     
         8 . The method of  claim 7  wherein the primary or secondary cell is selected from the group consisting of: fibroblasts, keratinocytes, epithelial cells, endothelial cells, glial cells, neural cells, formed elements of the blood, muscle cells, hepatocytes and precursors thereof.  
     
     
         9 . The method of  claim 1  further comprising culturing a homologously recombinant primary or secondary cell under conditions appropriate for propagating the homologously recombinant primary or secondary cell, thereby producing a clonal strain of homologously recombinant secondary cells.  
     
     
         10 . A homologously recombinant primary or secondary cell produced by the method of  claim 1 .  
     
     
         11 . A clonal cell strain of homologously recombinant secondary cells produced by the method of  claim 9 .  
     
     
         12 . A method of targeting DNA sequences into genomic DNA of a primary or secondary cell of vertebrate origin, comprising the steps of: 
 a) providing a DNA construct comprising: 
 1) exogenous DNA encoding a product to be expressed in primary or secondary cells of vertebrate origin;  
 2) DNA sequences homologous with genomic DNA sequences in the primary or secondary cell of vertebrate origin; and  
 3) DNA sequences encoding at least one selectable marker;  
   b) transfecting primary or secondary cells with the DNA construct provided in (a), thereby producing primary or secondary cells containing the DNA construct provided in (a); and    c) maintaining primary or secondary cells produced in (b) under conditions appropriate for homologous recombination to occur between DNA sequences homologous with genomic DNA sequences and genomic DNA sequences,    thereby producing homologously recombinant primary or secondary cells of vertebrate origin having the DNA construct of (a) integrated into genomic DNA of the primary or secondary cells.    
     
     
         13 . The method of  claim 12  wherein the primary or secondary cell is selected from the group consisting of: fibroblasts, keratinocytes, epithelial cells, endothelial cells, glial cells, neural cells, formed elements of the blood, muscle cells, hepatocytes and precursors thereof.  
     
     
         14 . The method of  claim 13  wherein the primary or secondary cell is of mammalian origin.  
     
     
         15 . The method of  claim 14  wherein the primary or secondary cell is selected from the group consisting of: primary human cells, secondary human cells, primary mouse cells, secondary mouse cells, primary rabbit cells and secondary rabbit cells.  
     
     
         16 . The method of  claim 15  wherein the exogenous DNA encodes a therapeutic product selected from the group consisting of: enzymes, cytokines, hormones, antigens, antibodies, clotting factors, regulatory proteins, ribozymes, transcription proteins, receptors, anti-sense nucleic acid sequences and novel proteins.  
     
     
         17 . The method of  claim 16  wherein the exogenous DNA is itself a therapeutic product selected from the group consisting of DNA sequences sufficient for sequestration of a protein or nucleic acid in the cell, DNA sequences which bind to a cellular regulatory protein, DNA sequences which alter secondary or tertiary chromosomal structure and DNA sequences which are transcriptional regulatory elements.  
     
     
         18 . A homologously recombinant primary or secondary cell produced by the method of  claim 12 .  
     
     
         19 . A homologously recombinant primary or secondary cell of vertebrate origin, having integrated into genomic DNA exogenous DNA.  
     
     
         20 . A homologously recombinant primary or secondary cell of  claim 19  selected from the group consisting of: fibroblasts, keratinocytes, epithelial cells, endothelial cells, glial cells, neural cells, formed elements of the blood, muscle cells, hepatocytes and precursors thereof.  
     
     
         21 - 64 . (Canceled).