DNA vector production system
AssigneeMediphage Bioceuticals, Inc.
InventorsRoderick Slavcev, Nafiseh Nafissi
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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.
Claims41
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, said expression vector comprising an e xpression cassette, and a nucleic acid of interest flanked on either side by a target binding sequence for the Tel recombinase and 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.
An expression vector adapted to produce a bacterial sequence-free vector, said expression vector comprising an expression cassette, and a nucleic acid of interest flanked on either side by a target binding sequence for the Tel recombinase and 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.
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, said expression vector comprising backbone sequence, an expression cassette, and a nucleic acid of interest flanked on either side by a target binding sequence for the Tel recombinase and 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.
A method of producing a bacterial sequence-free vector having covalently closed ends comprising:
- incubating a vector production system comprising recombinant cells designed to encode at least a first recombinase under control of an inducible promoter and to encode a nuclease genome editing system, under suitable conditions for expression of the first recombinase and the nuclease genome editing system,
- wherein the cells comprise an expression vector for producing the bacterial sequence-free vector having covalently closed ends, the expression vector comprising:
- a backbone sequence containing a cleavage site for the nuclease genome editing system,
- 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.
Dependencies are inferred from the claim text.