Patent ID: 8945885
Filing Date: 2015-02-03
Classification: A61K,C07K,C12N

Abstract:
1. A method for preparing a minicircle nucleic acid vector composition substantially free of contaminating nucleic acids, the method comprising: transfecting a genetically modified bacterial cell wherein the genetically modified bacterial cell genome comprises a disruption in an endA gene encoding endonuclease I and comprises a coding sequence for araE under control of a constitutive promoter, with a circular parental plasmid comprising: (i) a polynucleotide of interest flanked by attB and attP recombination sites recognized by a unidirectional site-specific recombinase; (ii) at least one restriction endonuclease site recognized by a restriction endonuclease not endogenous to the bacterial cell; wherein present in said circular parental plasmid or said bacterial cell are sequences encoding the unidirectional site-specific recombinase and the restriction endonuclease not endogenous to the bacterial cell; incubating the bacterial cells under conditions and for a period of time sufficient to express the unidirectional site-specific recombinase and allow the unidirectional site-specific recombinase to recombine the attB and attP recombination sites; and to express the restriction endonuclease and allow the restriction endonuclease to digest the restriction endonuclease site, wherein the incubating provides a minicircle nucleic acid vector comprising the polynucleotide of interest and a product hybrid sequence of the unidirectional site-specific recombinase; purifying the minicircle nucleic acid vector to provide a minicircle nucleic acid vector composition substantially free of contaminating nucleic acids comprising the nucleic acid coding sequence for the unidirectional site-specific recombinase and/or a restriction endonuclease.