Transient activation of transgene expression by hydrodynamics-based injection may cause rapid decrease in plasmid DNA expression

Abstract

The intranuclear disposition of exogenous DNA is quite important for the therapeutic effects of the administered DNA. The expression efficiency from one copy of exogenous DNA delivered by hydrodynamics-based injection dramatically decreases over time, and this 'silencing' occurs without CpG methylation. In this study, naked luciferase-plasmid DNA was delivered into mouse liver by hydrodynamics-based injection, and modifications of the histones bound to the plasmid DNA were analyzed by a chromatin immunoprecipitation (ChIP) analysis. In addition, the effects of a second hydrodynamics-based injection on the expression from the plasmid DNA were examined.The ChIP analysis revealed that the modification status of histone H3 remained constant from 4 h to 4 weeks. Surprisingly, the injection of saline without DNA enhanced the luciferase expression from the preexisting DNA administered 4 and 14 days previously. Our results suggest that histone modification plays no role in the silencing. Instead, our data suggest that the transgene expression is activated by the hydrodynamics-based injection manipulation, and that the return from the activated status causes the silencing.

Transgene expression efficiency from plasmid DNA delivered as a complex with histone H3

“…Therefore, histones could be a key factor for the intranuclear disposition of the exogenous DNA. In agreement with this hypothesis, transgene expression is affected by the introduction of DNA sequences with high and low histone affinities into plasmid DNAs (Nishikawa et al, 2003;Sumida et al, 2006;Kamiya et al, 2007Kamiya et al, , 2009.…”

“…The intranuclear disposition of the delivered plasmid is an important factor in efficient transgene expression (Kamiya et al, 2003). Nucleosomes are formed on non-integrated plasmid DNAs (Reeves et al, 1985), and the introduction of high and low histone-affinity DNA sequences into plasmid DNAs influences transgene expression (Nishikawa et al, 2003;Sumida et al, 2006;Kamiya et al, 2007Kamiya et al, , 2009. Moreover, decreased expression efficiency (silencing) seems to be accompanied by increased histone binding, not by histone modification (Ochiai et al, 2007).…”

“…Nucleosomes are formed on non-integrated plasmid DNAs (Reeves et al, 1985), and the introduction of high and low histone-affinity DNA sequences into plasmid DNAs influences transgene expression (Nishikawa et al, 2003;Sumida et al, 2006;Kamiya et al, 2007Kamiya et al, , 2009. Moreover, decreased expression efficiency (silencing) seems to be accompanied by increased histone binding, not by histone modification (Ochiai et al, 2007). Therefore, the control of the interaction(s) between the plasmid DNA and the histones seems to be one of the keys for controlling the intranuclear disposition.…”

Targeted sequence alteration of a chromosomal locus in mouse liver

“…The efficient transgene expression is due to effective delivery into hepatocytes (Liu et al, 1999), although activation of transgene by this injection procedure itself is another reason (Ochiai et al, 2007;Nishikawa et al, 2008). Moreover, efforts have been made toward establishing a hydrodynamic procedure for clinical use (Suda and Liu, 2007).…”

Inhibition of nuclear delivery of plasmid DNA and transcription by interferon γ: hurdles to be overcome for sustained gene therapy

“…The pellet was washed twice with the lysis buffer, and the final pellet was used as the nuclear fraction. 41,42 The intranuclear DNA was extracted using a DNeasy Blood & Tissue Kit (QIAGEN GmbH, Hilden, Germany). Total concentrations of DNA were determined by measurement of the absorbance at 260 nm in a UV-1600 spectrophotometer (Shimadzu, Kyoto, Japan).…”