Novel cho integration sites and uses thereof

Abstract

Expression-enhancing nucleotide sequences for eukaryotic expressions systems are provided that allow for enhanced and stable expression of recombinant proteins in eukaryotic cells. Genomic integration sites providing enhanced expression and methods of use thereof are provided for expression of a gene of interest in a eukaryotic cell. Chromosomal loci, sequences, and vectors are provided for enhanced and stable expression of genes in eukaryotic cells.

1 .- 55 . (canceled) 
     
     
         56 . A vehicle for modifying a CHO cell genome, comprising a vector that comprises:
 a. a 5′ homology arm homologous to a sequence present in the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4,   b. an exogenous nucleic acid, and   c. a 3′ homology arm homologous to a sequence present in the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4.   
     
     
         57 . The vehicle of  claim 56 , wherein the exogenous nucleic acid sequence comprises one or more recombination recognition sequences. 
     
     
         58 . The vehicle of  claim 57 , wherein the exogenous nucleic acid sequence comprises at least two recombination recognition sequences and a selectable marker placed between the two recombination recognition sequences. 
     
     
         59 . The vehicle of  claim 56 , wherein the exogenous nucleic acid sequence comprises a first exogenous gene of interest (GOI) and a first exogenous promoter, wherein the first exogenous GM is operably linked to the first exogenous promoter. 
     
     
         60 . The vehicle of  claim 59 , wherein the exogenous nucleic acid sequence further comprises a second exogenous GOI and a second exogenous promoter, wherein the second exogenous GOI is operably linked to a second exogenous promoter. 
     
     
         61 . The vehicle of  claim 60 , wherein the first exogenous GOI encodes a light chain of an antibody or antigen-binding fragment thereof, and the second exogenous (101 encodes a heavy chain of an antibody or antigen-binding fragment thereof. 
     
     
         62 . The vehicle of  claim 61 , wherein the exogenous nucleic acid sequence further comprises a third exogenous GOI and a third exogenous promoter, wherein the third exogenous GOI is operably linked to the third exogenous promoter. 
     
     
         63 . The vehicle of  claim 62 , wherein the first, second and third GOI encode a first light chain of an antibody or antigen-binding fragment thereof, a second light chain of an antibody or antigen-binding fragment thereof, and a heavy chain of an antibody or antigen-binding fragment thereof, respectively; or wherein the first, second and third GOI encode a light chain of an antibody or antigen-binding fragment thereof, a first heavy chain of an antibody or antigen-binding fragment thereof, and a second heavy chain of an antibody or antigen-binding fragment thereof, respectively. 
     
     
         64 . The vehicle of  claim 56 , wherein the 5′ homology arm comprises a nucleotide sequence present in the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO: 4 and is contiguous with the exogenous nucleic acid, and wherein the 3′ homology arm comprises a sequence present in the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO: 4 and is contiguous with the exogenous nucleic acid. 
     
     
         65 . The vehicle of  claim 56 , wherein the vehicle comprises at least an additional vector or an mRNA molecule. 
     
     
         66 . The vehicle  claim 65 , wherein the additional vector is selected from the group consisting of an adenovirus, a lentivirus, a retrovirus, an adeno-associated virus, an integrating phage vector, a non-viral vector, a transposon and/or transposase, an integrase substrate, and a plasmid. 
     
     
         67 . The vehicle of  claim 57 , wherein the vehicle comprises at least an additional vector which comprises a nucleic acid encoding a site-specific nuclease for integrating the recognition sequence, wherein the site-specific nuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a ZFN dimer, a transcription activator-like effector nuclease (TALEN), a TAL effector domain fusion protein, or an RNA-guided DNA endonuclease. 
     
     
         68 . A method of modifying the genome of a CHO cell, comprising:
 introducing a vehicle according to  claim 56  into a CHO cell, and integrating the exogenous nucleic acid into a locus in the CHO cell genome, wherein the locus comprises the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO: 4.   
     
     
         69 . A method, comprising:
 introducing into a CHO cell an exogenous nucleic acid, wherein the exogenous nucleic acid integrates into a locus of the genome, the locus comprising the nucleotide sequence of SEQ NO:1 or SEQ ID NO: 4, wherein the exogenous nucleic acid comprises a first exogenous gene of interest (GOI) and a first exogenous promoter, wherein the first exogenous GOI is operably linked to the first exogenous promoter.   
     
     
         70 . The method of  claim 69 , wherein the exogenous nucleic acid further comprises a second exogenous GOI and a second exogenous promoter, wherein the second exogenous GOI is operably linked to a second exogenous promoter. 
     
     
         71 . The method of  claim 70 , wherein the first exogenous GOI encodes a light chain of an antibody or antigen-binding fragment thereof, and the second exogenous GOI encodes a heavy chain of an antibody or antigen-binding fragment thereof. 
     
     
         72 . The method of  claim 69 , wherein the exogenous nucleic acid is introduced using a nucleic acid vector comprising:
 a. a 5′ homology arm homologous to a sequence present in the nucleotide sequence of SEQ NO:1 or SEQ II) NO: 4,   b. the exogenous nucleic acid, and   c. a 3′ homology arm homologous to a sequence present in the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO: 4.   
     
     
         73 . The method of  claim 72 , wherein the exogenous nucleic acid is introduced using at least an additional nucleic acid vector or an snRNA molecule. 
     
     
         74 . The method of  claim 73 , wherein the additional vector is selected from the group consisting of an adenovirus, a lentivirus, a retrovirus, an adeno-associated virus, an integrating phage vector, a non-viral vector, a transposon and/or transposase, an integrase substrate, and a plasmid. 
     
     
         75 . The method of  claim 73 , wherein the additional vector comprises a nucleic acid encoding a site-specific nuclease selected from the group consisting of a zinc finger nuclease (ZFN), a ZFN dimer, a transcription activator-like effector nuclease (TALEN), a TAL effector domain fusion protein, and an RNA-guided DNA endonuclease. 
     
     
         76 . The method of 69, wherein prior to introducing the exogenous nucleic acid, the CHO cell comprises one or more recombination recognition sequences integrated in said locus, and wherein the exogenous nucleic acid integrates into the one or more recombination recognition sequences in said locus. 
     
     
         77 . The method of  claim 76 , wherein prior to introducing the exogenous nucleic acid, the CHO cell comprises at least two recombination recognition sequences integrated in said locus, and a selectable marker placed between the two recombination recognition sequences. 
     
     
         78 . The method of  claim 77 , wherein the exogenous nucleic acid comprises a first exogenous gene of interest (GOI) operably linked to a first exogenous promoter, a first recombination recognition sequence placed at 5′ of the first exogenous promoter, and a second recombination recognition sequence placed at 3′ of the first exogenous GOI, and wherein the first and second recombination recognition sequences are identical, respectively, to the at least two recombination recognition sequences present in the locus of the genome prior to introducing the exogenous nucleic acid. 
     
     
         79 . The method of  claim 78 , wherein the exogenous nucleic acid further comprises a second exogenous GOI operably linked to a second exogenous promoter, wherein the second exogenous GOI is placed 3′ of the first exogenous GOI and 5′ of the second recombination recognition sequence 
     
     
         80 . The method of  claim 79 , wherein the first exogenous GOI encodes a light chain of an antibody or an antigen binding fragment thereof, and the second exogenous GOI encodes a heavy chain of an antibody or an antigen-binding fragment thereof or wherein the first exogenous GOI encodes a heavy chain of an antibody or antigen binding fragment thereof, and the second exogenous GOI encodes a light chain of an antibody or an antigen-binding fragment thereof.