Cell engineering using MICRORNAs
AssigneeBoehringer Ingelheim International GmbH
InventorsLore Florin, Hitto Kaufman, Angelika Hausser, Monilola Olayioye, Michaela Strotbek
Life of this patent
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Abstract
The invention concerns the field of cell culture technology. It concerns RNA having a specific sequence, expression vectors encoding the RNA, production host cell lines comprising the RNA, and methods of producing recombinant biopharmaceutical products using engineered host cell with altered levels of the 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 the RNAs. Those cell lines have improved secretion and/or growth characteristics in comparison to control cell lines.
Claims22
A method of developing a stably transfected mammalian cell comprising the following steps:
- (a) transfecting the mammalian cell with an expression vector encoding a selectable marker and at least one 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,
- (b) cultivating said cell for an initial period of time in the presence of selective pressure, and
- (c) selecting the transfected cell.
A method of producing a protein of interest, comprising the following steps:
- (a) transfecting at least one 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, or an expression vector encoding said at least one microRNA, and at least one expression vector comprising at least one gene of interest encoding a protein of interest into a mammalian cell,
- (b) selecting a highly-productive transfected cell producing the protein of interest, wherein the production and/or secretion of the protein of interest is increased compared to a control cell, which is not transfected with said at least one microRNA or the expression vector encoding said at least one microRNA, and
- (c) cultivating the highly-productive transfected cell obtained in step (b) under conditions which allow expression of the gene(s) of interest.
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 or genes that encode at least a product of interest and a selectable marker, wherein the cell is co-transfected with at least one 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, or an expression vector encoding said at least one microRNA,
- (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.
Dependencies are inferred from the claim text.