An araC-controlled Bacterialcre Expression System to Produce DNA Minicircle Vectors for Nuclear and Mitochondrial Gene Therapy

Abstract

The presence of CpG motifs and their associated sequences in bacterial DNA causes an immunotoxic response following the delivery of these plasmid vectors into mammalian hosts. We describe a biotechnological approach to the elimination of this problem by the creation of a bacterial cre recombinase expression system, tightly controlled by the arabinose regulon. This permits the Cre-mediated and -directed excision of the entire bacterial vector sequences from plasmid constructs to create supercoiled gene expression minicircles for gene therapy. Minicircle yields using standard culture volumes are sufficient for most in vitroand in vivo applications whereas minicircle expressionin vitro is significantly increased over standard plasmid transfection. By the simple expedient of removing the bacterial DNA complement, we significantly reduce the size and CpG content of these expression vectors, which should also reduce DNA-induced inflammatory responses in a dose-dependent manner. We further describe the generation of minicircle expression vectors for mammalian mitochondrial gene therapy, for which no other vector systems currently exist. The removal of bacterial vector sequences should permit appropriate transcription and correct transcriptional cleavage from the mitochondrial minicircle constructs in a mitochondrial environment and brings the realization of mitochondrial gene therapy a step closer. The presence of CpG motifs and their associated sequences in bacterial DNA causes an immunotoxic response following the delivery of these plasmid vectors into mammalian hosts. We describe a biotechnological approach to the elimination of this problem by the creation of a bacterial cre recombinase expression system, tightly controlled by the arabinose regulon. This permits the Cre-mediated and -directed excision of the entire bacterial vector sequences from plasmid constructs to create supercoiled gene expression minicircles for gene therapy. Minicircle yields using standard culture volumes are sufficient for most in vitroand in vivo applications whereas minicircle expressionin vitro is significantly increased over standard plasmid transfection. By the simple expedient of removing the bacterial DNA complement, we significantly reduce the size and CpG content of these expression vectors, which should also reduce DNA-induced inflammatory responses in a dose-dependent manner. We further describe the generation of minicircle expression vectors for mammalian mitochondrial gene therapy, for which no other vector systems currently exist. The removal of bacterial vector sequences should permit appropriate transcription and correct transcriptional cleavage from the mitochondrial minicircle constructs in a mitochondrial environment and brings the realization of mitochondrial gene therapy a step closer. base pair(s) synthetic ornithine transcarbamylase gene kilobase(s) Dulbecco's modified Eagle's medium fetal calf serum polymerase chain reaction cytomegalovirus left element loxP site right element loxPsite wild type There is increasing evidence to suggest that plasmid DNA used for non-viral gene delivery can cause unacceptable inflammatory responses in eukaryotes (1Krieg A.M. J. Lab. Clin. Med. 1996; 128: 128-133Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 2Yew N.S. Wang K.X. Przybylska M. Bagley R.G. Stedman M. Marshall J. Scheule R.K. Cheng S.H. Hum. Gene Ther. 1999; 10: 223-234Crossref PubMed Scopus (184) Google Scholar, 3Norman J. Denham W. Denham D. Yang J. Carter G. Abouhamze A. Tannahill C.L. MacKay S.L. Moldawer L.L. Gene Ther. 2000; 7: 1425-1430Crossref PubMed Scopus (40) Google Scholar, 4McLachlan G. Stevenson B.J. Davidson D.J. Porteous D.J. Gene Ther. 2000; 7: 384-392Crossref PubMed Scopus (66) Google Scholar, 5Krieg A.M. J. Gene Med. 1999; 1: 56-63Crossref PubMed Scopus (104) Google Scholar). These immunotoxic responses are largely due to the presence of unmethylated CpG motifs and their associated stimulatory sequences on plasmids, following bacterial propagation of plasmid DNA. Simple methylation of DNA in vitro may be enough to reduce an inflammatory response but is likely to result in severely depressed gene expression (6Krieg A.M. Mol. Ther. 2000; 1: 209-210Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The removal of CpG islands by cloning out, or elimination of non-essential sequences is more successful in reducing inflammatory responses but is time-consuming and tedious (7Yew N.S. Zhao H. Wu I.H. Song A. Tousignant J.D. Przybylska M. Cheng S.H. Mol. Ther. 2000; 1: 255-262Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar).Because bacterial DNA contains on average four times more CpG islands than does mammalian DNA (8Swartz M.N.T., T.A. Kornberg A. J. Biol. Chem. 1962; 237: 1961-1967Abstract Full Text PDF PubMed Google Scholar), a good solution is to entirely eliminate the bacterial control regions from gene delivery vectors during the process of plasmid production.Removal of bacterial sequences needs to be efficient, using the smallest possible excision site, while creating supercoiled DNA minicircles, consisting solely of gene expression elements under appropriate mammalian control regions.This can be achieved by the use of Cre recombinase, a bacteriophage P1-derived integrase Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Mol. Biol. PubMed Scopus Google Scholar), of base the of a supercoiled plasmid DNA by in the Cre DNA that are and supercoiled J. Biol. Chem. Full Text PDF PubMed Google Scholar), a minicircle the use of a bacterial cre expression for of supercoiled DNA. currently bacterial sufficient control of cre recombinase expression to during the bacterial and plasmid 1996; PubMed Scopus Google to Cre in of the minicircle due to by the and bacterial W. W. the tightly controlled arabinose expression for M. and and for to create bacterial which is and by the for by these minicircle we the of reaction by of the H. J. 7: PubMed Scopus Google Scholar, M. PubMed Scopus Google to a in reaction increased of This also to reduce from of minicircle approach to bacterial DNA from delivery vectors is also by on the of expression vectors for use in mitochondrial gene therapy. is to an ornithine transcarbamylase gene modified for mitochondrial 1996; PubMed Scopus Google Scholar). no vectors for mammalian mitochondrial gene we the gene the entire mitochondrial into a bacterial plasmid vector for propagation 1996; PubMed Scopus Google M. PubMed Google Scholar). to the of sequences mammalian the presence of a bacterial vector is likely to be to or of the of mitochondrial and of the bacterial vector sequences should this problem and reduce the size of these vectors, increasing the of their into this we describe the and of a bacterial tightly controlled and expression We this for DNA minicircle generation using a of for and mitochondrial gene expression from to We also the use of to the Cre reaction in yields of supercoiled significantly increased gene expression in vitro of this over standard plasmid and from from and bacterial from and A. a from D. and and 2000; PubMed Scopus Google Scholar). and used to a DNA loxP to create plasmid to the to create and used to create a for the plasmid and used to create and to create to a in and to of the used to a of into the bacterial constructs over the regions and gene expression loxP using the on a of the by the excision of of the and most of the This the control regions from the of the the and the also using to create of the bacterial and the into the bacterial gene using the of M. J. PubMed Google to of and loxP site from and loxP into the plasmid by and of the and the plasmid and to create The by and to a by the of and the loxP of to create of and loxP by the of from into the site of loxP and into by and further and to create The from and to the loxP by the of the entire plasmid removal of the of the using and to create by the of the from the from of by the of into the site of 2000; PubMed Scopus Google Scholar). The vector from this by and to by of site of 2000; PubMed Scopus Google Scholar). by the of the and most of the the of and Gene to the the appropriate minicircle to for in on in and in a minicircle plasmid a culture in of The most successful and cre in a in modified the of and for in of modified and in the of cre and further in a for in and for in of and in the of cre and further in a for of in in of solution by in and in of The and the DNA solution further by in and 1: Scholar). This yields of DNA of culture of DNA and bacterial vector the for and for mitochondrial supercoiled minicircle be from plasmid and bacterial vector on a using the W. J. more of achieved by in volumes of and in J. Scholar). Minicircle DNA to or to the to DNA from the of and into a culture in of medium fetal calf serum and of DNA in of to in of in the of of of to the this reaction and the to for in and a reaction of DNA in the in of and the the of the and following and of and using the Gene on a to the The in a using the to the of to that for of on the from for for of by the of This in that are more used the of to the for the and used a for on the which is more than on or the that are this a for the the and the smallest and this to the to a of The of is of the in this over a of the that no of are is describe the creation of a bacterial recombinase under the control of which can be used to of DNA minicircle in We also a of minicircle constructs for mitochondrial expression and for we and increased expression from minicircle constructs over for minicircle A.M. D. J. Gene Ther. PubMed Scopus Google Scholar, A.M. J. D. Gene Ther. 1999; PubMed Scopus Google Scholar, A.M. D. J. PubMed Scopus Google Scholar), used bacterial to minicircle DNA. This in excision of following A. PubMed Google Scholar). By the Cre-mediated in a site of Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Mol. Biol. PubMed Scopus Google Scholar), a of over of minicircle of culture are sufficient for most in vitro and in whereas further and of the process likely to be mitochondrial minicircles eliminate bacterial sequences that may be to mitochondrial of Google Scholar), or for we be that a loxP site into gene transcription and of these constructs in the mitochondrial constructs and by gene for the minicircle constructs from these are of a size that should their into J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We are currently the and of these mitochondrial minicircle vectors and the of the size of their plasmid these constructs increased over their plasmid on a and on a The in the to the of these vectors in reducing the of to while the of transcriptional by the simple expedient of removing the entire bacterial DNA complement, we also the CpG content of most of these expression vectors by more than minicircle expression vectors are likely to a for reducing inflammatory responses in non-viral vector vivo the increased in There is increasing evidence to suggest that plasmid DNA used for non-viral gene delivery can cause unacceptable inflammatory responses in eukaryotes (1Krieg A.M. J. Lab. Clin. Med. 1996; 128: 128-133Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 2Yew N.S. Wang K.X. Przybylska M. Bagley R.G. Stedman M. Marshall J. Scheule R.K. Cheng S.H. Hum. Gene Ther. 1999; 10: 223-234Crossref PubMed Scopus (184) Google Scholar, 3Norman J. Denham W. Denham D. Yang J. Carter G. Abouhamze A. Tannahill C.L. MacKay S.L. Moldawer L.L. Gene Ther. 2000; 7: 1425-1430Crossref PubMed Scopus (40) Google Scholar, 4McLachlan G. Stevenson B.J. Davidson D.J. Porteous D.J. Gene Ther. 2000; 7: 384-392Crossref PubMed Scopus (66) Google Scholar, 5Krieg A.M. J. Gene Med. 1999; 1: 56-63Crossref PubMed Scopus (104) Google Scholar). These immunotoxic responses are largely due to the presence of unmethylated CpG motifs and their associated stimulatory sequences on plasmids, following bacterial propagation of plasmid DNA. Simple methylation of DNA in vitro may be enough to reduce an inflammatory response but is likely to result in severely depressed gene expression (6Krieg A.M. Mol. Ther. 2000; 1: 209-210Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The removal of CpG islands by cloning out, or elimination of non-essential sequences is more successful in reducing inflammatory responses but is time-consuming and tedious (7Yew N.S. Zhao H. Wu I.H. Song A. Tousignant J.D. Przybylska M. Cheng S.H. Mol. Ther. 2000; 1: 255-262Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). bacterial DNA contains on average four times more CpG islands than does mammalian DNA (8Swartz M.N.T., T.A. Kornberg A. J. Biol. Chem. 1962; 237: 1961-1967Abstract Full Text PDF PubMed Google Scholar), a good solution is to entirely eliminate the bacterial control regions from gene delivery vectors during the process of plasmid of bacterial sequences needs to be efficient, using the smallest possible excision site, while creating supercoiled DNA minicircles, consisting solely of gene expression elements under appropriate mammalian control This can be achieved by the use of Cre recombinase, a bacteriophage P1-derived integrase Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Mol. Biol. PubMed Scopus Google Scholar), of base the of a supercoiled plasmid DNA by in the Cre DNA that are and supercoiled J. Biol. Chem. Full Text PDF PubMed Google Scholar), a minicircle the use of a bacterial cre expression for of supercoiled DNA. currently bacterial sufficient control of cre recombinase expression to during the bacterial and plasmid 1996; PubMed Scopus Google Scholar). This to Cre in of the minicircle due to by the and bacterial W. W. We the tightly controlled arabinose expression for M. and and for to create bacterial which is and by the for by these minicircle we the of reaction by of the H. J. 7: PubMed Scopus Google Scholar, M. PubMed Scopus Google to a in reaction increased of This also to reduce from of minicircle DNA. This approach to bacterial DNA from delivery vectors is also by on the of expression vectors for use in mitochondrial gene therapy. is to an ornithine transcarbamylase gene modified for mitochondrial 1996; PubMed Scopus Google Scholar). no vectors for mammalian mitochondrial gene we the gene the entire mitochondrial into a bacterial plasmid vector for propagation 1996; PubMed Scopus Google M. PubMed Google Scholar). to the of sequences mammalian the presence of a bacterial vector is likely to be to or of the of mitochondrial and of the bacterial vector sequences should this problem and reduce the size of these vectors, increasing the of their into this we describe the and of a bacterial tightly controlled and expression We this for DNA minicircle generation using a of for and mitochondrial gene expression from to We also the use of to the Cre reaction in yields of supercoiled significantly increased gene expression in vitro of this over standard plasmid and from from and bacterial from and A. a from D. and and 2000; PubMed Scopus Google Scholar). and used to a DNA loxP to create plasmid to the to create and used to create a for the plasmid and used to create and to create to a in and to of the used to a of into the bacterial constructs over the regions and gene expression loxP using the on a of the by the excision of of the and most of the This the control regions from the of the the and the also using to create of the bacterial and the into the bacterial gene using the of M. J. PubMed Google to of and loxP site from and loxP into the plasmid by and of the and the plasmid and to create The by and to a by the of and the loxP of to create of and loxP by the of from into the site of loxP and into by and further and to create The from and to the loxP by the of the entire plasmid removal of the of the using and to create by the of the from the from of by the of into the site of 2000; PubMed Scopus Google Scholar). The vector from this by and to by of site of 2000; PubMed Scopus Google Scholar). by the of the and most of the the of and Gene to the the appropriate minicircle to for in on in and in a minicircle plasmid a culture in of The most successful and cre in a in modified the of and for in of modified and in the of cre and further in a for in and for in of and in the of cre and further in a for of in in of solution by in and in of The and the DNA solution further by in and 1: Scholar). This yields of DNA of culture of DNA and bacterial vector the for and for mitochondrial supercoiled minicircle be from plasmid and bacterial vector on a using the W. J. more of achieved by in volumes of and in J. Scholar). Minicircle DNA to or to the to DNA from the of and into a culture in of medium fetal calf serum and of DNA in of to in of in the of of of to the this reaction and the to for in and a reaction of DNA in the in of and the the of the and following and of and using the Gene on a to the The in a using the to the of to that for of on the from for for of by the of This in that are more used the of to the for the and used a for on the which is more than on or the that are this a for the the and the smallest and this to the to a of The of is of the in this over a of the that no of are is and from from and bacterial from and A. a from D. and and 2000; PubMed Scopus Google Scholar). and used to a DNA loxP to create plasmid to the to create and used to create a for the plasmid and used to create and to create to a in and to of the used to a of into the bacterial constructs over the regions and gene expression loxP using the on a from from and bacterial from and A. a from D. and and 2000; PubMed Scopus Google Scholar). and used to a DNA loxP to create plasmid to the to create and used to create a for the plasmid and used to create and to create to a in and to of the used to a of into the bacterial constructs over the regions and gene expression loxP using the on a of the by the excision of of the and most of the This the control regions from the of the the and the also using to create by the excision of of the and most of the This the control regions from the of the the and the also using to create of the bacterial and the into the bacterial gene using the of M. 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This yields of DNA of culture of DNA and bacterial vector the for and for mitochondrial supercoiled minicircle be from plasmid and bacterial vector on a using the W. J. more of achieved by in volumes of and in J. Scholar). Minicircle DNA to or to the to DNA from the in and for in of and in the of cre and further in a for of in in of solution by in and in of The and the DNA solution further by in and 1: Scholar). This yields of DNA of culture The of DNA and bacterial vector the for and for mitochondrial supercoiled minicircle be from plasmid and bacterial vector on a using the W. J. more of achieved by in volumes of and in J. Scholar). Minicircle DNA to or to the to DNA from the of and into a culture in of medium fetal calf serum and of DNA in of to in of in the of of of to the this reaction and the to for in and a reaction of DNA in the in of and the the of the and following and into a culture in of medium fetal calf serum and of DNA in of to in of in the of of of to the this reaction and the to for in and a reaction of DNA in the in of and the the of the and following and of and using the Gene on a to the The in a using the to the of to that for of on the from for for of by the of This in that are more used the of to the for the and used a for on the which is more than on or the that are this a for the the and the smallest and this to the to a of The of is of the in this over a of the that no of are is using the Gene on a to the The in a using the to the of to that for of on the from for for of by the of This in that are more We used the of to the for the and used a for on the which is more than on or the that are this a for the the and the smallest and this to the to a of The of is of the in this over a of the that no of are is describe the creation of a bacterial recombinase under the control of which can be used to of DNA minicircle in We also a of minicircle constructs for mitochondrial expression and for we and increased expression from minicircle constructs over for minicircle A.M. D. J. Gene Ther. PubMed Scopus Google Scholar, A.M. J. D. Gene Ther. 1999; PubMed Scopus Google Scholar, A.M. D. J. PubMed Scopus Google Scholar), used bacterial to minicircle DNA. This in excision of following A. PubMed Google Scholar). By the Cre-mediated in a site of Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Mol. Biol. PubMed Scopus Google Scholar), a of over of minicircle of culture are sufficient for most in vitro and in whereas further and of the process likely to be mitochondrial minicircles eliminate bacterial sequences that may be to mitochondrial of Google Scholar), or for we be that a loxP site into gene transcription and of these constructs in the mitochondrial constructs and by gene for the minicircle constructs from these are of a size that should their into J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We are currently the and of these mitochondrial minicircle vectors and the of the size of their plasmid these constructs increased over their plasmid on a and on a The in the to the of these vectors in reducing the of to while the of transcriptional by the simple expedient of removing the entire bacterial DNA complement, we also the CpG content of most of these expression vectors by more than minicircle expression vectors are likely to a for reducing inflammatory responses in non-viral vector vivo the increased in We describe the creation of a bacterial recombinase under the control of which can be used to of DNA minicircle in We also a of minicircle constructs for mitochondrial expression and for we and increased expression from minicircle constructs over for minicircle A.M. D. J. Gene Ther. PubMed Scopus Google Scholar, A.M. J. D. Gene Ther. 1999; PubMed Scopus Google Scholar, A.M. D. J. PubMed Scopus Google Scholar), used bacterial to minicircle DNA. This in excision of following A. PubMed Google Scholar). By the Cre-mediated in a site of Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Mol. Biol. PubMed Scopus Google Scholar), a of over of minicircle of culture are sufficient for most in vitro and in whereas further and of the process likely to be The mitochondrial minicircles eliminate bacterial sequences that may be to mitochondrial of Google Scholar), or for we be that a loxP site into gene transcription and of these constructs in the mitochondrial constructs and by gene for the minicircle constructs from these are of a size that should their into J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We are currently the and of these mitochondrial The minicircle vectors and the of the size of their plasmid these constructs increased over their plasmid on a and on a The in the to the of these vectors in reducing the of to while the of transcriptional by the simple expedient of removing the entire bacterial DNA complement, we also the CpG content of most of these expression vectors by more than minicircle expression vectors are likely to a for reducing inflammatory responses in non-viral vector vivo the increased in We and A. for the of and bacterial D. and the expression plasmid to