US2015140591A1PendingUtilityA1
CELL ENGINEERING USING RNAs

Assignee: BOEHRINGER INGELHEIM INTPriority: Jun 6, 2012Filed: Jun 4, 2013Published: May 21, 2015
Est. expiryJun 6, 2032(~5.9 yrs left)· nominal 20-yr term from priority
Inventors:Lore Florin Hitto Kaufman Angelika Hausser Monilola Olayioye Michaela Strotbek
C12P 21/02C12N 15/113C12P 21/00C12N 15/111C12N 2330/50C07K 16/2884C07K 16/2887C12Y 111/01007C12N 9/0065C12N 2310/141C07K 14/62
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References
Claims
Abstract

The invention concerns the field of cell culture technology. It concerns RNA having a specific sequence, expression vectors encoding said RNA, production host cell lines comprising said RNA, and methods of producing recombinant biopharmaceutical products using engineered host cell with altered levels of said RNAs, such as small non-coding RNAs, preferably microRNAs (miRNAs). The invention also relates to engineered host cells with altered levels in one or more of said RNAs. Those cell lines have improved secretion and/or growth characteristics in comparison to control cell lines.
Claims

exact text as granted — not AI-modified
1 . A ribonucleic acid (RNA) selected from the group consisting of: SEQ ID NO:6, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, wherein said RNA leads to an increase in the production and/or secretion of a therapeutic protein of interest from a mammalian cell. 
     
     
         2 . A mammalian expression vector comprising a RNA selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. 
     
     
         3 . The mammalian expression vector of  claim 2 , wherein the vector comprises a polynucleotide sequence encoding the RNA. 
     
     
         4 . The mammalian expression vector of  claim 2 , wherein said RNA leads to an increase in the production and/or secretion of a therapeutic protein of interest in a mammalian expression system. 
     
     
         5 . The mammalian expression vector of  claim 4 , wherein either the protein of interest or the RNA is operably linked to an amplifiable selection marker. 
     
     
         6 . The mammalian expression vector of  claim 2 , further comprising a selection marker gene. 
     
     
         7 . The mammalian expression vector of  claim 6 , wherein the selection marker gene is an amplifiable selection marker gene. 
     
     
         8 . The mammalian expression vector of  claim 7 , wherein the amplifiable selection marker gene is a glutamine synthetase gene or a dihydrofolate reductase gene. 
     
     
         9 . The mammalian expression vector of  claim 2 , wherein
 the RNA is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.   
     
     
         10 . The mammalian expression vector according to  claim 2 , characterised in that it comprises a combination of several identical or different RNAs selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. 
     
     
         11 . The mammalian expression vector of  claim 10 , comprising a polynucleotide sequence encoding a combination of two or more of the RNAs, wherein the RNAs are identical or different. 
     
     
         12 . The mammalian expression vector according to  claim 10 , wherein the RNAs
 are selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.   
     
     
         13 . The mammalian expression vector of  claim 2 , further comprising at least one gene of interest. 
     
     
         14 . The mammalian expression vector of  claim 2 , wherein the RNA is a non-coding RNA. 
     
     
         15 . The mammalian expression vector of  claim 14 , wherein the non-coding RNA is a miRNA. 
     
     
         16 . A mammalian cell expression vector comprising a polynucleotide sequence encoding any of the RNAs of  claim 1 . 
     
     
         17 . The mammalian expression vector of  claim 16 , comprising a polynucleotide sequence encoding a combination of two or more of the RNAs of  claim 1 , wherein the RNAs are identical or different. 
     
     
         18 . A mammalian cell comprising one or more RNAs selected from one or more of the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. 
     
     
         19 . The mammalian cell of  claim 18 , wherein the cell is stably transfected with an expression vector encoding said one or more RNA. 
     
     
         20 . The mammalian cell of  claim 18 , wherein the one or more RNAs are non-coding RNAs. 
     
     
         21 . The mammalian cell of  claim 20 , wherein the non-coding RNAs are miRNAs. 
     
     
         22 . The mammalian cell of  claim 18 , wherein the RNA
 is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.   
     
     
         23 . The mammalian cell of  claim 18 , comprising a mammalian expression vector comprising a polynucleotide sequence encoding a RNA selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. 
     
     
         24 . The mammalian cell of  claim 18 , further stably expressing a protein of interest. 
     
     
         25 . The mammalian cell of  claim 18 , wherein the cell is a rodent or a human cell. 
     
     
         26 . The mammalian cell of  claim 25 , wherein the rodent cell is a hamster cell, preferably a CHO cell, most preferably a CHO DG44 cell. 
     
     
         27 . The mammalian cell of  claim 25 , wherein the human cell is a HEK-293 cell, a PER.C6 cell or a CAP cell. 
     
     
         28 . A method of developing a stably transfected mammalian cell comprising the following steps:
 (a) transfecting at least one RNA selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20 into the mammalian cell,   (b) cultivating said cell for an initial period of time in the presence of selective pressure, and   (c) selecting a high-producing transfected cell.   
     
     
         29 . The method of  claim 28 , wherein the mammalian cell in step (a) is stably transfected with an expression vector encoding said RNA. 
     
     
         30 . The method of  claim 28 , wherein the mammalian cell in step (a) is transfected with an expression vector comprising a polynucleotide sequence encoding a RNA selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. 
     
     
         31 . The method of  claim 28 , wherein the mammalian cell in step (a) is a producer host cell additionally comprising at least one expression vector comprising at least one gene of interest. 
     
     
         32 . A method of producing a protein of interest, comprising the following steps:
 (a) transfecting at least one RNA selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20 and at least one expression vector comprising at least one gene of interest into a mammalian cell,   (b) selecting a highly-productive transfected cell, and   (c) cultivating the highly-productive transfected cell obtained in step (b) under conditions which allow expression of the gene(s) of interest.   
     
     
         33 . The method of  claim 32 , further comprising a step (d) of harvesting and purifying the protein of interest 
     
     
         34 . The method of  claim 32 , wherein step (a) comprises transfecting an expression vector encoding said RNA. 
     
     
         35 . The method of  claim 34 , wherein the mammalian cell in step (a) is stably transfected with the expression vector encoding said RNA. 
     
     
         36 . The method of  claim 32 , wherein the mammalian cell in step (a) is transfected with an expression vector comprising a polynucleotide sequence encoding a RNA selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. 
     
     
         37 . The method of  claim 32 , wherein the expression vector comprising at least one gene of interest of step (a) also encodes the at least one RNA of step (a). 
     
     
         38 . The method of  claim 32 , wherein transfecting the RNA is done after, prior to or simultaneously to transfecting the gene of interest. 
     
     
         39 . A method of preparing and selecting a recombinant mammalian cell, comprising the following steps:
 (a) transfecting a mammalian cell with genes that encode at least a protein/product of interest and a selectable marker, wherein the cell is co-transfected with at least one RNA selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20,   (b) selecting a cell with co-integrated genes by cultivating the cell in the presence of a selective agent, and   (c) cultivating the cell under conditions which enable expression of the genes.   
     
     
         40 . The method of  claim 39  wherein the selectable marker confers resistance to neomycin, puromycin, bleomycin, zeozine or blasticidin. 
     
     
         41 . The method of  claim 39 , wherein the selectable marker is an amplifiable selectable marker and further comprising an additional step (b′) between steps (b) and (c), comprising amplifying the co-integrated genes by cultivating the cell in the presence of an agent which allows the amplification of the amplifiable selectable marker gene. 
     
     
         42 . The method of  claim 41 , wherein the amplifiable selectable marker gene encodes the amplifiable selectable markers dihydrofolate reductase or glutamine synthetase. 
     
     
         43 . The method of  claim 39 , wherein the mammalian cell in step (a) is stably transfected with an expression vector encoding said at least one RNA. 
     
     
         44 . The method of  claim 43 , wherein the mammalian cell in step (a) is transfected with an expression vector comprising a polynucleotide sequence encoding a RNA selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. 
     
     
         45 . The method of  claim 43 , wherein the expression vector encoding said at least one RNA is transfected after, prior to or simultaneously to transfecting the gene of interest. 
     
     
         46 . The method of  claim 32 , wherein the protein of interest is a recombinant therapeutic protein. 
     
     
         47 . The method of  claim 32 , wherein the protein of interest is an antibody or a Fc-fusion protein. 
     
     
         48 . The method of  claim 28 , wherein the production and/or secretion of the protein of interest is increased by 10%, 20%, 50%, 100%, 200%, 400% compared to a control cell, which is not transfected with at least one RNA selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. 
     
     
         49 . The method of  claim 28 , wherein the RNA is a non-coding RNA. 
     
     
         50 . The method of  claim 49 , wherein the non-coding RNA is a miRNA. 
     
     
         51 . (canceled) 
     
     
         52 . (canceled) 
     
     
         53 . (canceled) 
     
     
         54 . (canceled) 
     
     
         55 . (canceled) 
     
     
         56 . (canceled) 
     
     
         57 . (canceled) 
     
     
         58 . The mammalian cell of  claim 26 , wherein the hamster cell is a CHO cell.
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