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US 2022/0033845 A1Published application
Expression vectors for eukaryotic expression systems
AssigneeMerck Sharp & Dohme Corp.
InventorsZhimei Du, Bo Jiang, Xiaoyan Tang
Life of this patent
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Abstract
The invention provides expression vectors for expressing recombinant proteins (e.g., biologics) in mammalian cells. Also provided are host cells comprising the expression vectors, methods of producing the recombinant proteins, and methods of propagating the expression vectors.
Claims20
1-71. (canceled)
An expression vector comprising:
- (a) a first expression cassette comprising the following elements in the order of upstream to downstream:
- a promoter operably linked to an insertion site for a gene of interest (GOI), an internal ribosome entry site (IRES), a polynucleotide encoding a eukaryotic selectable marker, and a polyadenylation (polyA) signal;
- (b) a second expression cassette comprising a polynucleotide encoding a bacterial selectable marker;
- and
- (c) a bacterial plasmid origin of replication;
- wherein optionally the first expression cassette further comprises one or more regulatory element;
- and
- wherein optionally the regulatory element is an enhancer, an insulator, a locus control region (LCR), a matrix attachment region (MAR), a scaffold attachment region (SAR), an expression augmenting sequence element (EASE), an adenovirus tripartite leader (TPL), or a ubiquitous chromatin opening element (UCOE).
5 more elements
An expression vector comprising:
- (a) a first expression cassette comprising the following elements in the order of upstream to downstream:
- a first insulator, an EASE, a promoter, a TPL, an insertion site for a GOI, an IRES, a polynucleotide encoding a eukaryotic selectable marker, a polyA signal, and a second insulator;
- (b) two ITR sequences flanking the first expression cassette;
- (c) a second expression cassette comprising a polynucleotide encoding a bacterial selectable marker;
- and
- (d) a bacterial plasmid origin of replication;
- wherein optionally the first expression cassette further comprises an enhancer;
- wherein optionally the enhancer is located between the EASE and the promoter;
- wherein optionally the enhancer is a human CMV immediate-early enhancer;
- wherein optionally the promoter is a human CMV immediate-early promoter;
- wherein optionally the first and the second insulators are HS4;
- wherein optionally the ITR is piggyBac ITR;
- wherein optionally the eukaryotic selectable marker is a glutamine synthetase or a neomycin phosphotransferase;
- and
- wherein optionally the IRES comprises a polynucleotide sequence of SEQ ID NO:1, 2, 3, 23, 24, or 25.
12 more elements
An expression vector,
- (a) comprising a polynucleotide sequence of SEQ ID NO:4, 5, 6, 7, 8, 26, 27, 28, or 29;
- (b) consisting of a polynucleotide sequence of SEQ ID NO:4, 5, 6, 7, 8, 26, 27, 28, or 29;
- or
- (c) consisting essentially of a polynucleotide sequence of SEQ ID NO:4, 5, 6, 7, 8, 26, 27, 28, or 29.
1 more element
Dependencies are inferred from the claim text.