DNA vector production system
AssigneeMediphage Bioceuticals, Inc.
InventorsRoderick Slavcev, Nafiseh Nafissi
Life of this patent
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Abstract
A vector production system is provided. The system comprises recombinant cells designed to encode at least a first recombinase under the control of an inducible promoter and the cells include an expression vector encoding a nucleic acid of interest within the regulatory elements of the expression vector which are flanked on either side by a target sequence for at least the first recombinase. The vector production system provides an efficient one-step process for producing linear or circular covalently closed vectors that incorporate a nucleic acid sequence of interest.
Claims25
A vector production system comprising recombinant cells designed to encode at least a first recombinase under the control of an inducible promoter, wherein said cells comprise an expression vector adapted to produce a bacterial sequence-free vector having covalently closed ends, said expression vector comprising:
- an expression cassette, and comprising a nucleic acid sequence of interest flanked on either side by,
- a target binding sequence for the Tel first recombinase and flanking each side of the expression cassette,
- one or more additional target sequences for one or more additional recombinases integrated within non-binding regions of the Tel target binding sequence are target binding sequences for one or more additional recombinases, wherein the nucleic acid of interest encodes a nuclease genome editing system for the first recombinase, and
- at least one enhancer sequence flanking each side of the target sequence for the first recombinase to facilitate nuclear uptake and enhance transfection efficiency of the bacterial sequence-free vector and expression of the nucleic acid sequence of interest.
An expression vector adapted to produce a bacterial sequence-free vector having covalently closed ends, said expression vector comprising:
- an expression cassette, and comprising a nucleic acid sequence of interest flanked on either side by,
- a target binding sequence for the Tel a first recombinase and flanking each side of the expression cassette,
- one or more additional target sequences for one or more additional recombinases integrated within non-binding regions of the Tel target binding sequence are target binding sequences for one or more additional recombinases, wherein the nucleic acid of interest encodes a nuclease genome editing system for the first recombinase, and
- at least one enhancer sequence flanking each side of the target sequence for the first recombinase to facilitate nuclear uptake and enhance transfection efficiency of the bacterial sequence-free vector and expression of the nucleic acid sequence of interest.
A vector production system comprising recombinant cells designed to encode at least a first recombinase under the control of an inducible promoter and to encode a nuclease genome editing system, wherein said cells comprise an expression vector adapted to produce a bacterial sequence-free vector having covalently closed ends, said expression vector comprising:
- a backbone sequence containing a cleavage site for the nuclease genome editing system,
- an expression cassette, and comprising a nucleic acid sequence of interest flanked on either side by,
- a target binding sequence for the Tel first recombinase and flanking each side of the expression cassette,
- one or more additional target sequences for one or more additional recombinases integrated within non-binding regions of the Tel target binding sequence are target binding sequences for one or more additional recombinases, wherein the cells are genetically modified to encode a nuclease genome editing system adapted to cleave the expression vector at a site within the backbone sequence for the first recombinase, and
- at least one enhancer sequence flanking each side of the target sequence for the first recombinase to facilitate nuclear uptake and enhance transfection efficiency of the bacterial sequence-free vector and expression of the nucleic acid sequence of interest.
A vector production system comprising recombinant cells designed to encode at least a first recombinase under the control of an inducible promoter, wherein said cells comprise an expression vector adapted to produce a bacterial sequence-free vector having covalently closed ends, said expression vector comprising:
- an expression cassette comprising a nucleic acid sequence of interest,
- a target sequence for the first recombinase flanking each side of the expression cassette, and
- one or more additional target sequences for one or more additional recombinases integrated within non-binding regions of the target sequence for the first recombinase,
- wherein the target sequence for the first recombinase and the one or more additional target sequences for the one or more additional recombinases have a total base pair length of less than or equal to 142 base pairs.
A vector production system comprising recombinant cells designed to encode at least a first recombinase under the control of an inducible promoter, wherein said cells comprise an expression vector adapted to produce a bacterial sequence-free vector having covalently closed ends, said expression vector comprising:
- an expression cassette comprising a nucleic acid sequence of interest,
- a target sequence for the first recombinase flanking each side of the expression cassette, and
- at least five additional target sequences for at least five additional recombinases integrated within non-binding regions of the target sequence for the first recombinase,
- wherein the target sequence for the first recombinase and the at least five additional target sequences for the at least five additional recombinases have a total base pair length of less than or equal to 300 base pairs.
Dependencies are inferred from the claim text.