Methods and compositions for expression of polypeptides in a cell
Inventors
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10538799-B2
Publication Date
2020-01-21
Expiration Date
Abstract
Disclosed herein are vector systems for expression of polypeptides in eukaryotic cells; and methods of obtaining high-level expression of polypeptides in a eukaryotic cell. Methods and compositions for obtaining stable, long-term expression of recombinant polypeptides are also provided.
Core Innovation
The invention provides viral-derived transcriptional control regions for recombinant polypeptide expression in eukaryotic cells, using a cytomegalovirus (CMV) major immediate early (MIE) promoter consisting of the nucleotide sequence of SEQ ID NO:3. The polynucleotide further includes a transgene, a polyadenylation signal, and a woodchuck hepatitis virus post transcriptional regulatory element (WPRE) consisting of the nucleotide sequence of SEQ ID NO:7.
The disclosed transcriptional control region architecture is described as a regulatory sequence configuration including RC-dUTR/promoter/uUTR/intron/dUTR with SEQ ID NOs 1-6, together with the WPRE element. The description further recites optional incorporation of post-transcriptional and structural regulatory elements, including MAR/SAR sequences, in combination with the CMV MIE promoter and WPRE.
Experimental results described in the disclosure indicate that incorporation of WPRE markedly increases the proportion of stable high-expressing clones/mini-pools. The disclosure also reports improved expression longevity, stated as up to about 50 generations, along with elevated expression levels for exemplary recombinant polypeptides, including rituximab light chain and rituximab heavy chain, and human growth hormone.
Claims Coverage
The independent claim recites a polynucleotide defined by a CMV MIE promoter (SEQ ID NO:3) together with a WPRE (SEQ ID NO:7), and includes a transgene and a polyadenylation signal. Additional inventive features recited across the claims include a multiple cloning site, MAR/SAR sequences, specified MAR/SAR sequence selection, transgene product categories, and a containing cell that is prokaryotic or eukaryotic.
CMV MIE promoter and WPRE transcriptional architecture
A polynucleotide comprising a cytomegalovirus (CMV) major immediate early (MIE) promoter consisting of the nucleotide sequence of SEQ ID NO:3, a transgene, a polyadenylation signal, and a woodchuck hepatitis virus post transcriptional regulatory element (WPRE) consisting of the nucleotide sequence of SEQ ID NO:7.
Multiple cloning site addition
The polynucleotide additionally includes a multiple cloning site.
MAR/SAR structural regulatory element inclusion
The claimed polynucleotide includes a matrix attachment region and/or a scaffold attachment region (MAR/SAR) sequence in addition to the features of claim 1.
Specified MAR/SAR sequence selection
The MAR/SAP sequence is chosen from specified chicken lysozyme and interferon-derived MAR/SAR sequences or hybrids thereof, including chicken lysozyme MAR/SAR (CLM), interferon alpha-2 MAR/SAR (IAM), interferon beta MAR/SAR (IBM), X29 MAR/SAR, and S4 MAR/SAR, or hybrids thereof.
Transgene product limited to protein categories
The polynucleotide is defined such that its transgene encodes a protein selected from recombinant proteins, fusion proteins, antibodies, cytokines, hormones, enzymes, or clotting factors.
Containing cell is prokaryotic or eukaryotic
A cell that contains the polynucleotide, where the cell is either prokaryotic or eukaryotic.
Overall claim coverage is centered on a polynucleotide construction combining a CMV MIE promoter defined by SEQ ID NO:3 with a WPRE defined by SEQ ID NO:7, together with a transgene and a polyadenylation signal. Dependent claim coverage further expands the construct with a multiple cloning site and MAR/SAR sequences, restricts MAR/SAR selection to specified sequences including hybrids, narrows the transgene product to particular protein categories, and optionally specifies the containing cell as prokaryotic or eukaryotic.
Stated Advantages
Increases the proportion of stable high-expressing clones/mini-pools.
Improves expression longevity, reported as up to about 50 generations.
Elevates expression levels for recombinant polypeptides.
Documented Applications
Expression of recombinant polypeptides including rituximab light chain and rituximab heavy chain.
Expression of human growth hormone.
Use in eukaryotic cells for recombinant polypeptide expression.
Interested in licensing this patent?