Abstract
We previously demonstrated that sustainable enhanced levels of transgene products could be expressed from a bacterial DNA-free expression cassette either formed from a fragmented plasmid in mouse liver or delivered as a minicircle vector. This suggested that bacterial DNA sequences played a role in episomal transgene silencing. To further understand the silencing mechanism, we systematically altered the DNA components in both the expression cassette and the bacterial backbone, and compared the gene expression profiles from mice receiving different DNA forms. In nine vectors tested, animals that received the purified expression cassette alone always expressed persistently higher levels of transgene compared to 2fDNA groups. In contrast, animals that received linearized DNA by a single cut in the bacterial backbone had similar expression profiles to that of intact plasmid groups. All three linear DNAs formed large concatemers and small circles in mouse liver, while ccDNA remained intact. In all groups, the relative amount of vector DNA in liver remained similar. Together, these results further established that the DNA silencing effect was mediated by a covalent linkage of the expression cassette and the bacteria DNA elements.
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References
Chen ZY et al. Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver. Mol Ther 2001; 3: 403–410.
Gill DR et al. Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1alpha promoter. Gene Therapy 2001; 8: 1539–1546.
Hartikka J et al. An improved plasmid DNA expression vector for direct injection into skeletal muscle. Hum Gene Ther 1996; 7: 1205–1217.
Nicol F et al. Poly-L-glutamate, an anionic polymer, enhances transgene expression for plasmids delivered by intramuscular injection with in vivo electroporation. Gene Therapy 2002; 9: 1351–1358.
Wolff JA et al. Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. Hum Mol Genet 1992; 1: 363–369.
Qin L et al. Promoter attenuation in gene therapy: interferon-gamma and tumor necrosis factor-alpha inhibit transgene expression. Hum Gene Ther 1997; 8: 2019–2029.
Yew NS et al. High and sustained transgene expression in vivo from plasmid vectors containing a hybrid ubiquitin promoter. Mol Ther 2001; 4: 75–82.
Li S et al. Effect of immune response on gene transfer to the lung via systemic administration of cationic lipidic vectors. Am J Physiol 1999; 276: L796–804.
Tan Y, Li S, Pitt BR, Huang L . The inhibitory role of CpG immunostimulatory motifs in cationic lipid vector-mediated transgene expression in vivo. Hum Gene Ther 1999; 10: 2153–2161.
Paillard F . CpG: the double-edged sword. Hum Gene Ther 1999; 10: 2089–2090.
Miao CH, Thompson AR, Loeb K, Ye X . Long-term and therapeutic-level hepatic gene expression of human factor IX after naked plasmid transfer in vivo. Mol Ther 2001; 3: 947–957.
Yew NS et al. CpG-depleted plasmid DNA vectors with enhanced safety and long-term gene expression in vivo. Mol Ther 2002; 5: 731–738.
Liu F, Song Y, Liu D . Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Therapy 1999; 6: 1258–1266.
Kramer MG et al. In vitro and in vivo comparative study of chimeric liver-specific promoters. Mol Ther 2003; 7: 375–385.
Critchlow SE, Jackson SP . DNA end-joining: from yeast to man. Trends Biochem Sci 1998; 23: 394–398.
Chen ZY, He CY, Ehrhrdt A, Kay MA . Minicircle DNA vectors devoid of bacterial DNA result in persistent and high level transgene expression in vivo. Mol Ther 2003; 8: 495–500.
Wolffe AP . Histone modification and transcriptional competence. In: Driel RV, Otte AP (eds) Nuclear Organization, Chromatin Structure, and Gene Expression. Oxford University Press: Oxford, New York, Tokyo, 1997, pp 40–57.
Utley RT et al. Transcriptional activators direct histone acetyltransferase complexes to nucleosomes. Nature 1998; 394: 498–502.
Noma K, Allis CD, Grewal SI . Transitions in distinct histone H3 methylation patterns at the heterochromatin domain boundaries. Science 2001; 293: 1150–1155.
Kass SU, Landsberger N, Wolffe AP . DNA methylation directs a time-dependent repression of transcription initiation. Curr Biol 1997; 7: 157–165.
Krieg AM et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 1995; 374: 546–549.
Krieg AM et al. Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs. Proc Natl Acad Sci USA 1998; 95: 12631–12636.
Schwartz DA et al. CpG motifs in bacterial DNA cause inflammation in the lower respiratory tract. J Clin Invest 1997; 100: 68–73.
Yew NS et al. Contribution of plasmid DNA to inflammation in the lung after administration of cationic lipid:pDNA complexes. Hum Gene Ther 1999; 10: 223–234.
Freimark BD et al. Cationic lipids enhance cytokine and cell influx levels in the lung following administration of plasmid: cationic lipid complexes. J Immunol 1998; 160: 4580–4586.
Scheule RK et al. Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung. Hum Gene Ther 1997; 8: 689–707.
Huang CY et al. Enhancements in gene expression by the choice of plasmid DNA formulations containing neutral polymeric excipients. J Pharmacol Sci 2002; 91: 1371–1381.
Hemmi H et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000; 408: 740–745.
Hong K, Sherley J, Lauffenburger DA . Methylation of episomal plasmids as a barrier to transient gene expression via a synthetic delivery vector. Biomol Eng 2001; 18: 185–192.
Holliday R, Pugh JE . DNA modification mechanisms and gene activity during development. Science 1975; 187: 226–232.
Sager R, Kitchin R . Selective silencing of eukaryotic DNA. Science 1975; 189: 426–433.
Bird AP, Wolffe AP . Methylation-induced repression – belts, braces, and chromatin. Cell 1999; 99: 451–454.
Schubeler D et al. Genomic targeting of methylated DNA: influence of methylation on transcription, replication, chromatin structure, and histone acetylation. Mol Cell Biol 2000; 20: 9103–9112.
Mummaneni P, Walker KA, Bishop PL, Turker MS . Epigenetic gene inactivation induced by a cis-acting methylation center. J Biol Chem 1995; 270: 788–792.
Miao CH et al. Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro. Mol Ther 2000; 1: 522–532.
Yant SR et al. Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nat Genet 2000; 25: 35–41.
Zhang G, Budker V, Wolff JA . High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum Gene Ther 1999; 10: 1735–1737.
Acknowledgements
We are extremely grateful to Theresa Storm for critical reading of this paper. This work was supported by NIH HL-64274.
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Chen, Z., He, C., Meuse, L. et al. Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo. Gene Ther 11, 856–864 (2004). https://doi.org/10.1038/sj.gt.3302231
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DOI: https://doi.org/10.1038/sj.gt.3302231
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