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-11692193-B2
Publication Date
2023-07-04
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 disclosure describes recombinant mammalian expression vectors that include a first and a second expression cassette arranged in tandem. Each cassette encodes an immunoglobulin subunit open reading frame operably linked to specified control sequences and followed by polyadenylation sites.
In the first expression cassette, a control sequence comprises a murine cytomegalovirus (mCMV) enhancer element together with a CMV promoter sequence at its 3′ end, with operable linkage 5′ to a rat EF-1alpha intron sequence. The first cassette further includes intervening and leader sequences operably linked relative to the CMV promoter sequence and the rat EF-1alpha intron, and a first polyadenylation site operably linked 3′ to the first immunoglobulin subunit open reading frame.
The vector includes a second expression cassette positioned 3′ to the first expression cassette, with a promoter control sequence operably linked to a second immunoglobulin subunit open reading frame and a second polyadenylation site operably linked 3′ to the second open reading frame. A transcription termination sequence is positioned 3′ to the first expression cassette and 5′ to the second expression cassette, and some embodiments further incorporate additional regulatory/selection components and genomic integration for stable expression in mammalian host cells, including CHO cells, with in vitro protein production and recovery.
Claims Coverage
The independent claim defines a recombinant expression vector with two tandem immunoglobulin subunit expression cassettes, defined enhancer/promoter-intron control architecture, leader and polyadenylation elements, and a transcription termination sequence positioned between the cassettes. Dependent claims refine additional regulatory/selection components, host-cell context, and an in vitro production and recovery use for a protein of interest.
Two tandem immunoglobulin expression cassettes with coordinated transcription termination
A recombinant expression vector comprising a first expression cassette and a second expression cassette arranged 3′ to the first expression cassette, where a transcription termination sequence is 3′ to the first expression cassette and 5′ to the second expression cassette.
mCMV enhancer and CMV promoter operably linked to rat EF-1alpha intron
A control sequence comprising a murine cytomegalovirus (mCMV) enhancer element with a CMV promoter sequence at its 3′ end, operably linked 5′ to a rat EF-1alpha intron sequence, with leader sequences operably linked relative to the CMV promoter sequence and the rat EF-1alpha intron sequence.
Immunoglobulin subunit open reading frames with operably linked polyadenylation sites
A first expression cassette comprising 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, and a second expression cassette comprising an open reading frame encoding a second immunoglobulin subunit operably linked to a promoter and a second polyadenylation site operably linked 3′ to the open reading frame.
Overall, the independent claim focuses on a recombinant expression vector configured with tandem expression cassettes for first and second immunoglobulin subunits, using a control sequence that couples an mCMV enhancer with a CMV promoter and operable linkage to a rat EF-1alpha intron, together with defined leader, polyadenylation, and transcription termination positioning.
Stated Advantages
The document describes modifications intended to reduce metabolic burden and improve producing pools.
Documented Applications
In vitro production and recovery of a protein of interest from a mammalian host cell culture medium.
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