Replicative minicircle vectors with improved expression
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Publication Number
US-10844388-B2
Publication Date
2020-11-24
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 ii) a 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 or 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 pUC-free eukaryotic replicative minicircle expression vectors that include a eukaryotic region sequence encoding a gene of interest and comprising 5′ and 3′ ends. A short spacer region having a length of less than 500 basepairs links the 5′ and 3′ ends, and the spacer comprises an R6K bacterial replication origin and an RNA selectable marker.
In particular, the R6K bacterial replication origin has at least 95% sequence identity to SEQ ID NO: 11. The RNA selectable marker is described as RNA-OUT, RNAI, or synthetic RNA selectable marker variants, including functional RNA-OUT embodiments with sequence identity constraints to the specified SEQ ID sequences.
The document describes insertion of the R6K/ColE2 replication origins and RNA selectable markers into eukaryotic Pol II units, including an intron or a 3′ UTR, and states that this does not reduce splicing/function and can increase transgene expression in vitro and markedly in vivo. It also emphasizes safety and manufacturing rationale, including conditional replication origin and elimination of protein/antibiotic markers.
Claims Coverage
The independent claims recite a pUC-free eukaryotic replicative minicircle expression vector with a short spacer (<500 basepairs) linking 5′ and 3′ ends, containing an R6K bacterial replication origin and an RNA selectable marker. The claims also require sequence identity constraints for the R6K origin, and dependent claims further define the RNA selectable marker embodiments, including functional RNA-OUT and RNAI or synthetic RNA selectable marker variants.
Puc-free eukaryotic replicative minicircle expression vector with short spacer
A eukaryotic replicative pUC-free minicircle expression vector comprising a eukaryotic region sequence encoding a gene of interest and comprising 5′ and 3′ ends, and a spacer region having a length of less than 500 basepairs that links the 5′ and 3′ ends and comprises an R6K bacterial replication origin and an RNA selectable marker.
R6K replication origin sequence identity constraint
The R6K bacterial replication origin has at least 95% sequence identity to SEQ ID NO: 11.
Functional RNA-OUT RNA selectable marker
The RNA selectable marker is a functional RNA-OUT having at least 95% sequence identity to a sequence selected from SEQ ID NO: 20 or SEQ ID NO: 22, and in one embodiment has at least 95% sequence identity to SEQ ID NO: 21.
RNA selectable marker selected among RNA-OUT, RNAI, or synthetic RNA selectable marker variants
The RNA selectable marker is selected from RNA-OUT, RNAI, or synthetic RNA selectable marker, each defined by encoded regulating RNAs having at least 95% sequence identity to listed SEQ ID sequences.
Overall, the claims cover a pUC-free eukaryotic replicative minicircle expression vector defined by a <500 bp spacer linking 5′ and 3′ ends, where the spacer contains an R6K bacterial replication origin with at least 95% identity to SEQ ID NO: 11 and an RNA selectable marker. The dependent claims further narrow the RNA selectable marker as functional RNA-OUT and/or RNAI or synthetic RNA selectable marker variants with specified sequence-identity constraints.
Stated Advantages
Does not reduce splicing/function when inserted into eukaryotic Pol II units such as an intron or a 3′ UTR.
Increased transgene expression in vitro and markedly in vivo.
Improved manufacturing due to removal of long (>500 bp) spacer bacterial regions, including high fermentation titers.
Improved in vivo expression.
Substantially improved manufacturing yields.
Safety/regulatory motivation through conditional replication origin.
Elimination of protein/antibiotic markers.
Documented Applications
Eukaryotic expression of a gene of interest using the described replicative minicircle/nanoplasmid vectors, including examples using an EGFP transgene and a muSEAP transgene.
Improved manufacturing in fermentation, including the stated example of HyperGRO fermentation with high fermentation titers.
Use of replicative minicircle elements inserted into Pol II units such as an intron or a 3′ UTR to maintain splicing/function and support transgene expression.
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