Inducible expression from transposon-based vectors and uses
Inventors
McGrew, Jeffrey T. • Smidt, Pauline S. • Ong, E-Ching
Assignees
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Publication Number
US-11685933-B2
Publication Date
2023-06-27
Expiration Date
Abstract
Recombinant expression vectors are disclosed that include a control sequence for recombinant expression of proteins of interest; the control sequence combines a mCMV enhancer sequence with a rat EF-1alpha intron sequence. Some of the vectors are useful for tetracycline-inducible expression. Some of the vectors contain a 5′ PiggyBac ITR and a 3′ PiggyBac ITR to promote genomic integration into a host cell chromosome. A method of selecting a stable production cell line for manufacturing a protein of interest is also disclosed. Also disclosed are mammalian host cells comprising the inventive recombinant expression vectors and a method of producing a protein of interest, in vitro, involving the mammalian host cell.
Core Innovation
The invention provides a recombinant expression vector with a first expression cassette and a second expression cassette, configured for expression of immunoglobulin subunits from operably linked regulatory elements. The control sequence for the first expression cassette includes a murine cytomegalovirus (mCMV) enhancer element together with a CMV promoter sequence at its 3′ end, and the CMV promoter sequence is operably linked 5′ to a rat EF-1α intron. Within the CMV promoter, one or more TetO sequences are inserted, and the control sequence includes an intervening first leader sequence and a second leader sequence arranged between the CMV promoter, the rat EF-1α intron, and the 3′ end of the control sequence.
The first expression cassette further includes an open reading frame encoding a first immunoglobulin subunit operably linked to the control sequence, and a first polyadenylation site operably linked 3′ to that open reading frame. The second expression cassette is positioned 3′ to the first expression cassette and includes a control sequence comprising a promoter, an open reading frame encoding a second immunoglobulin subunit operably linked to the promoter, and a second polyadenylation site operably linked 3′ to the second immunoglobulin subunit open reading frame. A transcription termination sequence is provided 3′ to the first expression cassette and 5′ to the second expression cassette.
The invention provides a PiggyBac transposon-based recombinant expression vector designed for stable integration in mammalian cells. The vector includes specified PiggyBac 5′ and 3′ inverted terminal repeats and expression cassettes configured for expression of a protein of interest from immunoglobulin subunits. TetR-mediated repression is used during selection in the absence of tetracycline, followed by derepression with tetracycline to enable expression for stable production.
Claims Coverage
The independent claim is a recombinant expression vector including two immunoglobulin-expression cassettes with a TetO-containing mCMV/CMV promoter-intron control sequence for the first subunit and a promoter-driven control sequence for the second subunit, together with polyadenylation and transcription termination elements. A PiggyBac transposon-based vector with 5′ and 3′ inverted terminal repeats is also described.
TetO-containing mCMV/CMV enhancer promoter linked to rat EF-1alpha intron
A control sequence comprising a murine cytomegalovirus (mCMV) enhancer sequence including an mCMV enhancer element (mCMV-E) and a CMV promoter (CMV-P) sequence at its 3′ end, operably linked 5′ to a rat EF-1alpha intron sequence, comprising one or more TetO sequences inserted within the CMV-P sequence.
Two-part leader arrangement flanking the CMV-P/intron control sequence
An intervening first leader sequence operably linked 3′ to the CMV-P sequence of the mCMV enhancer sequence and 5′ to the rat EF-1alpha intron sequence, and a second leader sequence operably linked 3′ to the rat EF-1alpha intron sequence.
First immunoglobulin subunit cassette with polyadenylation
An open reading frame encoding a first immunoglobulin subunit operably linked to the control sequence, and a first polyadenylation site operably linked 3′ to the open reading frame encoding the first immunoglobulin subunit.
Second immunoglobulin subunit cassette positioned 3′ with promoter and polyadenylation
A second expression cassette 3′ to the first expression cassette, comprising a control sequence comprising a promoter, an open reading frame encoding a second immunoglobulin subunit operably linked to the promoter, and a second polyadenylation site operably linked 3′ to the open reading frame encoding the second immunoglobulin subunit.
Transcription termination positioned between and beyond cassettes
A transcription termination sequence 3′ to the first expression cassette and 5′ to the second expression cassette.
PiggyBac transposon-based recombinant expression vector
A PiggyBac transposon-based recombinant expression vector with PiggyBac 5′ and 3′ inverted terminal repeats designed for stable integration in mammalian cells.
Overall, claim coverage centers on a recombinant expression vector architecture with two immunoglobulin-expression cassettes, specified expression-control elements, polyadenylation and transcription termination elements, and PiggyBac inverted terminal repeats. The first cassette uses an mCMV enhancer/CMV promoter linked to a rat EF-1α intron with one or more TetO sequences inserted within the CMV-P sequence.
Stated Advantages
TetR-mediated repression is used during selection in the absence of tetracycline, followed by derepression with tetracycline to enable expression for stable production.
Designed for stable integration in mammalian cells.
Documented Applications
Expression of immunoglobulin subunits.
Expression of a protein of interest from immunoglobulin subunits.
Host cell and in vitro protein production and recovery concepts using culture medium.
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