Replicative minicircle vectors with improved expression
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Publication Number
US-11851665-B2
Publication Date
2023-12-26
Expiration Date
Abstract
A eukaryotic replicative pUC-free minicircle expression vector is provided. The eukaryotic replicative pUC-free minicircle expression vector includes a pUC-free eukaryotic region sequence encoding a transgene of interest and comprising 5′ and 3′ ends and a ii) pUC-free spacer region of less than 500 basepairs in length linking the 5′ and 3′ ends of the eukaryotic region sequences and comprising a bacterial R6K replication origin having at least 95% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 12 and a RNA selectable marker, the RNA selectable marker being an RNA-IN regulating RNA-OUT functional variant having at least 95% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 22.
Core Innovation
The invention provides a eukaryotic replicative pUC-free minicircle expression vector. The vector includes a eukaryotic region sequence encoding a gene of interest and comprising 5′ and 3′ ends, and the eukaryotic region is linked by a spacer region that connects the 5′ and 3′ ends.
The spacer region comprises a R6K bacterial replication origin having at least 95% sequence identity to SEQ ID NO:11. The disclosed vectors also include an RNA selectable marker, including RNA-OUT and RNA-IN regulating RNA-OUT functional variants, and support insertion of intronic and 3′UTR regions.
The document describes increased plasmid encoded transgene expression, accurate intron splicing and EGFP expression, improved in vivo expression compared with plasmid and minicircle comparators, and improved duration of expression. It also states improved fermentation/manufacturing yields without complex spacer-region removal.
Claims Coverage
The provided claim material centers on a pUC-free minicircle expression vector with a spacer-linked R6K bacterial replication origin having at least 95% sequence identity to SEQ ID NO:11. Dependent claims add an RNA selectable marker, including a functional RNA-OUT with defined sequence identity references.
Eukaryotic region encoding gene of interest with 5′ and 3′ ends
A eukaryotic replicative pUC-free minicircle expression vector comprising a eukaryotic region sequence encoding a gene of interest and comprising 5′ and 3′ ends.
Spacer region linking 5′ and 3′ ends
A spacer region that links the 5′ and 3′ ends of the eukaryotic region sequence.
R6K bacterial replication origin with at least 95% identity to SEQ ID NO: 11
The spacer region comprises a R6K bacterial replication origin, wherein the R6K bacterial replication origin comprises a nucleic acid sequence with at least 95% sequence identity to SEQ ID NO: 11.
RNA selectable marker addition
The vector further includes an RNA selectable marker.
Functional RNA-OUT with identity to SEQ ID NO: 20 or SEQ ID NO: 22
The RNA selectable marker is a functional RNA-OUT having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 20 or SEQ ID NO: 22.
Overall, the claim coverage centers on a pUC-free minicircle expression vector with a spacer-linked R6K bacterial replication origin. Additional coverage is provided by an RNA selectable marker, including embodiments where the marker is a functional RNA-OUT with specified identity references.
Stated Advantages
Increased plasmid encoded transgene expression.
Accurate intron splicing and EGFP expression.
Improved in vivo expression compared with plasmid and minicircle comparators.
Improved duration of expression.
Substantially improved fermentation/manufacturing yields.
Improved expression and manufacturing without complex spacer-region removal.
Reduced safety concerns compared to protein or antibiotic markers.
Documented Applications
Expression of a gene of interest in eukaryotic contexts, including in vivo expression in BALB/c mice and expression testing in A549 and HEK293 cell lines.
In vivo transgene expression using the disclosed eukaryotic replicative pUC-free minicircle expression vectors.
Fermentation/manufacturing of the vector for improved yields.
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