Regulatory Nucleic Acid Elements

Abstract

The invention relates to DNA-sequences, especially transcription- or expression-enhancing elements (TE elements) and their use on an expression vector in conjunction with an enhancer, a promoter, a product gene and a selectable marker. The invention describes Sequence No. 1 and TE elements TE-01, -02, -03, -04, -06, -07, -08, -10, -11 or -12. Because of their small size, TE-06, TE-07 or TE-08 are particularly preferred. Sequence No. 1 originates from a sequence region located upstream from the coding region of the Ub/S27 a gene from CHO cells. TE elements bring about an increase in the expression of the product gene, particularly when stably integrated in the eukaryotic genome, preferably the CHO-DG44 genome. Chromosomal positional effects are thereby overcome, shielded or cancelled out. In this way the proportion of high producers in a transfection mixture and also the absolute expression level are increased up to seven-fold.

1 . A nucleic acid comprising TE-13 (SEQ ID No. 15) or a fragment of TE-13 (SEQ ID No. 15) or the complementary nucleotide sequences thereof or a derivative of TE-13 (SEQ ID No. 15) or a fragment thereof or the complementary nucleotide sequences thereof, wherein on chromosomal integration said nucleic acid leads to an increase in the transcription or expression of a gene of interest in an expression system. 
     
     
         2 . The nucleic acid according to  claim 1 , wherein said nucleic acid comprises TE-08 (SEQ ID No. 10) or a fragment of TE-08 (SEQ ID No. 10) or the complementary nucleotide sequences thereof or a derivative of TE-08 (SEQ ID No. 10) or a fragment thereof or the complementary nucleotide sequences thereof, wherein on chromosomal integration said nucleic acid leads to an increase in the transcription or expression of a gene of interest in an expression system. 
     
     
         3 . The nucleic acid according to  claim 2 , wherein said nucleic acid comprises SEQ ID No. 1 or a fragment of SEQ ID No. 1 or the complementary nucleotide sequences thereof or a derivative of SEQ ID No. 1 or a fragment thereof or the complementary nucleotide sequences thereof, wherein on chromosomal integration said nucleic acid leads to an increase in the transcription or expression of a gene of interest in an expression system, wherein said fragment comprises at least one sequence region from the nucleic acid region between 1 bp and 1578 bp (in relation to SEQ ID No. 1). 
     
     
         4 . The nucleic acid according to  claim 1 , wherein the increase in the transcription or expression of a gene of interest in an expression system in comparison to a control which does not contain a TE element, is determined by measuring the product titre by ELISA. 
     
     
         5 . The nucleic acid according to  claim 1 , wherein said nucleic acid hybridises under stringent conditions
 (a) with the region of nucleic acid sequence TE-13 (SEQ ID No. 15); or   (b) the complementary nucleic acid sequences thereof, or   (c) a nucleic acid sequence which has at least about 70% sequence identity with a sequence of (a) or (b).   
     
     
         6 . The nucleic acid according to  claim 5 , wherein said nucleic acid has a length of at least 1015 bp (=length TE-8, SEQ ID No. 10). 
     
     
         7 . The nucleic acid according to  claim 5 , wherein said nucleic acid has a length of at least 511 bp (=length TE-13, SEQ ID No. 15). 
     
     
         8 . The nucleic acid according to  claim 2 , wherein said nucleic acid hybridises under stringent conditions
 (a) with the region of nucleic acid sequence TE-08 (SEQ ID No. 10); or   (b) the complementary nucleic acid sequences thereof, or   (c) a nucleic acid sequence which has at least about 70% sequence identity with a sequence of (a) or (b).   
     
     
         9 . The nucleic acid according to  claim 1 , wherein said nucleic acid is a fragment or derivative of TE-01 (SEQ ID No. 3). 
     
     
         10 . The nucleic acid according to  claim 9 , wherein said nucleic acid is TE-13 (SEQ ID No. 15), TE-14 (SEQ ID No. 16), TE-15 (SEQ ID No. 17), TE-16 (SEQ ID No. 18), TE-17 (SEQ ID No. 19) or TE-18 (SEQ ID No. 20). 
     
     
         11 . The nucleic acid according to  claim 1 , wherein said nucleic acid or a fragment or derivative thereof is an isolated nucleic acid. 
     
     
         12 . The nucleic acid according to  claim 1 , wherein said nucleic acid is linked to a heterologous sequence. 
     
     
         13 . An isolated nucleic acid selected from the group consisting of TE-00 (SEQ ID No. 2), TE-01 (SEQ ID No. 3), TE-02 (SEQ ID No. 4), TE-03 (SEQ ID No. 5), TE-04 (SEQ ID No. 6), TE-06 (SEQ ID No. 8), TE-07 (SEQ ID No. 9), TE-08 (SEQ ID No. 10), TE-10 (SEQ ID No. 12), TE-11 (SEQ ID No. 13), TE-12 (SEQ ID No. 14), TE-13 (SEQ ID No. 15), TE-14 (SEQ ID No. 16), TE-15 (SEQ ID No. 17), TE-16 (SEQ ID No. 18), TE-17 (SEQ ID No. 19), TE-18 (SEQ ID No. 20) and TE-21 (SEQ ID No. 21). 
     
     
         14 . The isolated nucleic acid according to  claim 13 , wherein said nucleic acid is TE-06 (SEQ ID No. 8). 
     
     
         15 . The isolated nucleic acid according to  claim 13 , wherein said nucleic acid is TE-08 (SEQ ID No. 10). 
     
     
         16 . The nucleic acid according to  claim 13 , wherein said nucleic acid is TE-13 (SEQ ID No. 15). 
     
     
         17 . A eukaryotic expression vector comprising a nucleic acid according to  claim 1 . 
     
     
         18 . The eukaryotic expression vector according to  claim 17 , wherein said expression vector further comprises a promoter and a heterologous gene of interest. 
     
     
         19 . The eukaryotic expression vector according to  claim 18 , wherein said expression vector further comprises an enhancer. 
     
     
         20 . The eukaryotic expression vector according to  claim 18 , wherein said expression vector further comprises a selectable marker. 
     
     
         21 . The eukaryotic expression vector according to  claim 20 , wherein said selectable marker is DHFR, Neo, or Neo F240I. 
     
     
         22 . The eukaryotic expression vector according to  claim 17 , wherein said expression vector comprises a combination of several identical or different said nucleic acids, wherein one or more said nucleic acids are positioned in front of (i.e. 5′ of) said gene of interest, or one or more said nucleic acids are positioned behind (i.e. 3′ of) said gene of interest, or one or more said nucleic acids are positioned in front of and behind said gene of interest. 
     
     
         23 . The eukaryotic expression vector according to  claim 22 , wherein said nucleic acids are TE-06 (SEQ ID No. 8), TE-21 (SEQ ID No. 21) or TE-08 (SEQ ID No. 10). 
     
     
         24 . The eukaryotic expression vector according to  claim 22 , wherein said combination comprises a TE-08 nucleic acid (SEQ ID No. 10) followed by a TE-06-nucleic acid (SEQ ID No. 8). 
     
     
         25 . The eukaryotic expression vector according to  claim 22 , said combination comprises one or more TE-08-nucleic acid(s) (SEQ ID No. 10) positioned in front of (i.e. 5′ of) and one or more TE-08-nucleic acid(s) (SEQ ID No. 10) positioned behind (i.e. 3′ of) said gene of interest. 
     
     
         26 . The eukaryotic expression vector according to one of  claim 17 , wherein said expression vector comprises one or more said nucleic acids distributed over 2 plasmids. 
     
     
         27 . Method of producing a eukaryotic expression vector comprising the step of integrating a nucleic acid according to  claim 1  in an expression vector. 
     
     
         28 . A eukaryotic host cell comprising a eukaryotic expression vector according to  claim 17 . 
     
     
         29 . The eukaryotic host cell according to  claim 28 , wherein said cell is a high producer, wherein said cell has a higher specific productivity than a comparable eukaryotic host cell lacking a TE element, wherein said host cell has an expression level which is increased up to two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold or ten-fold or one which is increased more than two-fold, more than three-fold, more than four-fold, more than five-fold, more than seven-fold or more than ten-fold when compared to said cell lacking a TE element. 
     
     
         30 . The eukaryotic host cell according to  claim 28 , wherein said expression vector is stably integrated in the genome of said cell. 
     
     
         31 . The eukaryotic host cell according to  claim 28  wherein said host cell is a mammalian cell. 
     
     
         32 . The eukaryotic host cell according to  claim 31 , wherein said host cell is a CHO, NSO, Sp2/0-Ag14, BHK21, BHK TK − , HaK, 2254-62.2 (BHK-21-derivative), CHO-K1, CHO-DUKX (═CHO duk − , CHO/dhfr − ), CHO-DUKX B1, CHO-DG44, CHO Pro-5, V79, B14AF28-G3, or CHL cell. 
     
     
         33 . The eukaryotic host cell according to  claim 32 , wherein said host cell is a CHO-DG44 cell. 
     
     
         34 . The eukaryotic host cell according to  claim 28 , wherein said cell further comprises an anti-apoptosis gene. 
     
     
         35 . The eukaryotic host cell according to  claim 34 , wherein said anti-apoptosis gene is BCL-xL, BCL-2, BCL-w, BFL-1, A1, MCL-1, BOO, BRAG-1, NR-13, CDN-1, CDN-2, CDN-3, BHRF-1, LMW5-HL or CED-9. 
     
     
         36 . Method of developing a high-producing stably transfected eukaryotic host cell line comprising the steps of:
 (a) integrating at least one nucleic acid according to  claim 1  in a eukaryotic expression vector containing a gene of interest,   (b) transfecting a eukaryotic host cell with an expression vector obtained in step (a),   (c) selecting a highly-productive transfected host cell.   
     
     
         37 . The method according to  claim 36 , further comprising an amplification step. 
     
     
         38 . The method according to  claim 37 , further comprising a cloning step. 
     
     
         39 . Method of preparing and selecting recombinant mammalian cells comprising the steps of:
 (a) transfecting the host cells with a gene that codes for a protein/product of interest, a neomycin-phosphotransferase, and the amplifiable selectable marker DHFR, wherein in order to enhance the transcription or expression said gene of interest is functionally linked to at least one nucleic acid according to  claim 1 ,   (b) cultivating the cells under conditions which enable expression of said genes,   (c) selecting these co-integrated genes by cultivating the cells in the presence of a selecting agent in a hypoxanthine/thymidine-free medium, and   (d) amplifying these co-integrated genes by cultivating the cells in the presence of a selecting agent which allows the amplification of at least the amplifiable selectable marker gene.   
     
     
         40 . The method according to  claim 39 , wherein said transfected cells are cultivated in hypoxanthine/thymidine-free medium, supplemented with at least 200 μg/mL G418, in the absence of serum and with the addition of increasing concentrations of methotrexate (MTX). 
     
     
         41 . The method according to  claim 40 , wherein the concentration of MTX in the first amplification step is at least 100 nM or at least 250 nM and is increased stepwise to 1 μM or above. 
     
     
         42 . The method according to  claim 39 , further comprising improving the glycosylation of said protein of interest. 
     
     
         43 . The method according to  claim 39 , wherein said host cell is a mammalian cell. 
     
     
         44 . The method according to  claim 43 , wherein said mammalian cell is a CHO, NS0, Sp2/0-Ag14, BHK21, BHK TK − , HaK, 2254-62.2 (BHK-21-derivative), CHO-K1, CHO-DUKX (═CHO duke, CHO/dhfr − ), CHO-DUKX B1, CHO-DG44, CHO Pro-5, V79, B14AF28-G3, CHL cell. 
     
     
         45 . The method according to  claim 44 , wherein said mammalian cell is a CHO-DG44 cell. 
     
     
         46 . The method according to  claim 39 , wherein the expression vector comprises the selectable marker DHFR. 
     
     
         47 . The method according to  claim 39 , wherein the proportion of high producers is increased up to two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold or ten-fold or more than two-fold, more than three-fold, more than four-fold, more than five-fold, more than seven-fold or more than ten-fold. 
     
     
         48 . Method of preparing a biopharmaceutical product comprising the steps of:
 (a) integrating at least one nucleic acid according to  claim 1  in a eukaryotic expression vector containing a gene of interest,   (b) transfecting a eukaryotic host cell with an expression vector obtained in step (a),   (c) selecting a highly-productive transfected host cell obtained in step (b) and   (d) cultivating the highly-productive transfected host cell selected in step (c) under conditions which allow expression of the gene(s) of interest.   
     
     
         49 . The method according to  claim 48 , further comprising the step of:
 (e) harvesting and purifying said protein of interest.   
     
     
         50 . Kit consisting of a nucleic acid according to  claim 1 , an expression vector and a host cell. 
     
     
         51 . The kit according to  claim 50 , further comprising a transfection reagent.