Replicative minicircle vectors with improved expression
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Publication Number
US-11098313-B2
Publication Date
2021-08-24
Expiration Date
Abstract
The present invention relates to the production and use of covalently closed circular (ccc) recombinant DNA molecules such as plasmids, cosmids, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors and hybrids thereof, and more particularly to vector modifications that improve expression of said DNA molecules.
Core Innovation
The invention relates to a eukaryotic replicative minicircle expression vector and minimalized eukaryotic expression plasmid that include a gene of interest eukaryotic region having 5′ and 3′ ends, an intron with a 5′ splice donor and a 3′ acceptor branch site, and a 3′ UTR. A spacer region of less than 500 basepairs links the 5′ and 3′ ends of the eukaryotic region sequences.
The vector includes a bacterial replication origin positioned at a first location selected from within the spacer region, within the 3′ UTR, and within the intron between the 5′ splice donor and the 3′ acceptor branch site. A RNA selectable marker is positioned at a second location selected from the same locations, with the first location different from the second location.
The bacterial replication origin is an R6K bacterial replication origin having at least 95% sequence identity to SEQ ID NO: 11. The document describes replicative minicircle and nanoplasmid configurations, including intron-embedded and/or 3′UTR-encoded bacterial replication origins and separate placement of replication origin and RNA selectable marker.
Claims Coverage
The document provides one independent claim defining a replicative minicircle expression vector with an intron/3′ UTR gene-of-interest region, a spacer region of less than 500 bp, and separate placement of a bacterial replication origin and an RNA selectable marker at distinct locations. The independent claim contains four key inventive features, including an R6K bacterial replication origin with at least 95% sequence identity to SEQ ID NO:11, while dependent claims refine RNA selectable marker types and intron sequence variants.
Eukaryotic region with intron splice signals and 3′ UTR
A eukaryotic region sequence having 5′ and 3′ ends encoding a gene of interest, the eukaryotic region sequence including an intron with a 5′ splice donor and a 3′ acceptor branch site and a 3′ UTR.
Short spacer region linking the 5′ and 3′ ends
A spacer region of less than 500 basepairs linking the 5′ and 3′ ends of the eukaryotic region sequences.
R6K bacterial replication origin at a selectable intron/spacer/3′UTR location
A bacterial replication origin positioned at a first location selected from within the spacer region, within the 3′ UTR, and within the intron between the 5′ splice donor and the 3′ acceptor branch site, where the bacterial replication origin is an R6K bacterial replication origin having at least 95% sequence identity to the sequence of SEQ ID NO: 11.
RNA selectable marker at a different intron/spacer/3′UTR location
An RNA selectable marker positioned at a second location selected from within the spacer region, within the 3′ UTR, and within the intron between the 5′ splice donor and the 3′ acceptor branch site, wherein the first location is different than the second location.
Overall claim coverage centers on a eukaryotic replicative minicircle expression vector in which a <500 bp spacer links 5′/3′ ends, an intron with defined splice elements and a 3′ UTR are included in the eukaryotic region, an R6K replication origin is placed at one selectable intron/spacer/3′UTR location, and an RNA selectable marker is placed at a different selectable location.
Stated Advantages
Improved plasmid/transgene expression.
Improved in vivo and in vitro EGFP/muSEAP expression, with reported fold increases.
Accurate splicing/export measurements.
High fermentation/plasmid yields with reduced manufacturing complexity versus minicircles requiring spacer-region removal.
Simplified and/or boosted manufacturing yields.
High manufacturing yields described as above 100 mg/L up to about 745 mg/L.
Documented Applications
Gene therapy.
Vaccination.
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