US2022064690A1PendingUtilityA1

CELL ENGINEERING USING RNAs

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Assignee: BOEHRINGER INGELHEIM INTPriority: Jun 6, 2012Filed: Nov 12, 2021Published: Mar 3, 2022
Est. expiryJun 6, 2032(~5.9 yrs left)· nominal 20-yr term from priority
Inventors:Lore FlorinHitto KaufmannAngelika HausserMonilola OlayioyeMichaela Strotbek
C12N 15/111C12Y 111/01007C12N 2330/50C07K 16/2884C12N 9/0065C12P 21/02C12N 2310/141C12N 15/113C12P 21/00C07K 14/62C07K 16/2887
67
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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 . (canceled) 
     
     
         2 . A mammalian expression vector comprising a microRNA 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 microRNA. 
     
     
         4 . The mammalian expression vector of  claim 2 , wherein said microRNA leads to an increase in the production and/or secretion of a therapeutic protein of interest in a mammalian expression system. 
     
     
         5 . (canceled) 
     
     
         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 microRNA is selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 11, and SEQ ID NO: 16. 
     
     
         10 . The mammalian expression vector according to  claim 2 , characterised in that it comprises a combination of several identical or different microRNAs 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 microRNAs, wherein the RNAs are identical or different. 
     
     
         12 . The mammalian expression vector according to  claim 10 , wherein the microRNAs are selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 11, and SEQ ID NO: 16. 
     
     
         13 . The mammalian expression vector of  claim 2 , further comprising at least one gene of interest. 
     
     
         14 - 17 . (canceled) 
     
     
         18 . A mammalian cell comprising one or more microRNAs 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, wherein the cell is stably transfected with an expression vector encoding said one or more microRNA. 
     
     
         19 - 21 . (canceled) 
     
     
         22 . The mammalian cell of  claim 18 , wherein the microRNA is selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 11, and SEQ ID NO: 16. 
     
     
         23 . The mammalian cell of  claim 18 , comprising a mammalian expression vector comprising a polynucleotide sequence encoding a microRNA 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. 
     
     
         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 - 51 . (canceled) 
     
     
         52 . The mammalian expression vector of  claim 2 , wherein the expression vector is a plasmid or a viral vector. 
     
     
         53 . The mammalian cell of  claim 18 , wherein the expression vector is a plasmid or a viral vector. 
     
     
         54 . A CHO cell comprising one or more microRNAs selected from the group consisting of: miR-1287 (SEQ ID NO:6), miR-1978 (SEQ ID NO:11), and miR-557 (SEQ ID NO:16). 
     
     
         55 . The CHO cell of  claim 54 , wherein the CHO cell further stably expresses a protein of interest. 
     
     
         56 . The CHO cell of  claim 55 , wherein the protein of interest is a recombinant protein. 
     
     
         57 . The CHO cell of  claim 54 , wherein the cell is stably transfected with an expression vector encoding said one or more microRNA.

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