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CpG-Free DNA
CpGs and the Immune Response
Bacterial DNA is rich in unmethylated 2’-deoxyribo (cytidine-phosphateguanosine)
(CpG) dinucleotides, in contrast to mammalian DNA which
contains a low frequency of CpG dinucleotides that are mostly
methylated. Unmethylated CpGs in specific sequence contexts activate
the vertebrate immune system via Toll-Like Receptor (TLR) 9. TLR9
recognizes CpG DNA and initiates a signaling cascade leading to the
production of proinflammatory cytokines such as IL-6 and IL-12 1 .
Plasmids used for in vivo experiments are produced in E. coli and therefore
their CpGs are unmethylated and induce immune responses through this
host defense mechanism, which represents a limitation for the clinical
development of DNA vaccines and gene therapy vectors.
CpGs and Gene Silencing
A major limitation of gene delivery vectors for gene therapy applications is
the rapid decline of transgene expression in vivo. Methylation of CpG
dinucleotides within the promoter is a major factor limiting long-lasting gene
expression. Indeed, the transcriptional activity of the widely used and CpGrich
cytomegalovirus immediate early gene promoter (CMV) is highly robust
but prone to inactivation within a few weeks 2 . Replacement of the CMV
promoter with a cellular promoter combined with the use of a CpG-reduced
backbone was shown by our team (figures 1 & 2) and other labs to increase
the duration of expression and lower the inflammatory response 3, 4 . Recently,
CpGs in plasmid DNA (pDNA) were found to enhance the clearance of
PEG-coated pDNA-lipoplexes, a phenomenon that could be reduced by the
use of CpG-free pDNA allowing repeated dosing 5 .
Construction of CpG-free Plasmids
InvivoGen has developed a new family of plasmids that are completely
devoid of CpG dinucleotides, named pCpGfree. pCpGfree plasmids
contain elements that either naturally lack CpG dinucleotides, were
modified to remove all CpGs, or entirely synthesized such as genes
encoding selectable markers or reporters. These plasmids yield high levels
of transgene expression both in vitro and in vivo, and in contrast to CMVbased
plasmids allow sustained expression in vivo (figure 1) 6 .
Applications of CpG-free Plasmids
A major application of CpG-free plasmids is the treatment of inherited
diseases cause by a single gene defect, such as cystic fibrosis and hemophilia,
through gene therapy. Hyde et al. have reported promising results for the
treatment of cystic fibrosis using a pCpGfree-derived plasmid 4 . They show
that a CpG-free pDNA can achieve sustained lung transgene expression in
contrast to a pDNA containing even a single CpG. These results have led
to a clinical trial that has started in February 2009.
pCpGfree plamids represent valuable tools to study the effects of CpGs on
gene expression using cell lines expressing TLR9 7 , as well as their effects on
the innate and acquired immune systems. Furthermore, pCpGfree plasmids
are also useful when analyzing promoter methylation 8, 9 .
1. Bauer S. et al., 2001. Human TLR9 confers responsiveness to bacterial DNA via speciesspecific
CpG motif recognition. PNAS USA. 98(16):9237-42. 2. Scharfmann R. et al., 1991.
Long-term in vivo expression of retrovirus-mediated gene transfer in mouse fibroblast implants.
PNAS U S A. 88(11):4626-30. 3. Yew NS. et al., 2001. High and sustained transgene expression
in vivo from plasmid vectors containing a hybrid ubiquitin promoter. Mol Ther. 4(1):75-82.
4. Hyde SC. et al., 2008. CpG-free plasmids confer reduced inflammation and sustained
pulmonary gene expression. Nat Biotechnol. 26(5):549-51. 5. Tagami T. et al., 2010. CpG motifs
in pDNA-sequences increase anti-PEG IgM production induced by PEG-coated pDNAlipoplexes.
J Control Release. 142(2):160-6 6. Hattori K. et al., 2010. Sustained Exogenous
Expression of Therapeutic Levels of IFN-{gamma} Ameliorates Atopic Dermatitis in NC/Nga
Mice via Th1 Polarization. J Immunol. 184(5):2729-35. 7.Yasuda K. et al., 2009. Requirement for
DNA CpG content in TLR9-dependent dendritic cell activation induced by DNA-containing
immune complexes. J Immunol. 183(5):3109-17. 8. Klug M & Rehli M., 2006. Functional analysis
of promoter CpG methylation using a CpG-free luciferase reporter vector. Epigenetics. 1(3):127-
30. 9. van Straten EM. et al., 2009. The Liver X-Receptor (LXR) gene promoter is
hypermethylated in a mouse model of prenatal protein restriction. Am J Physiol Regul Integr
Comp Physiol. 282(2):R275-82.
Luciferase (μg/ml)
Figure 1. Time course of luciferase expression. Plasmids (30 μg) expressing a CpGfree
luciferase (ssLuc) gene and containing different amounts of CpGs in their
backbone were hydrodynamically injected in Swiss mice. Luciferase expression was
evaluated at different time points. Expression of ssLuc from pcDNA3-CMV (CpGrich
plasmid with CMV promoter) peaked at day 1 after injection but had drastically
fallen at day 4 to reach background levels by day 8. In contrast, ssLuc expression
from pCpGfree-SLS (CpG-free plasmid with a CpG-free engineered albumin
promoter) was high and persisted to day 47. Rapid reduction of luciferase
expression was also observed with pCpGfree-CMV (CpG-free plasmid with CMV
promoter) and pcDNA3-SLS (CpG-rich plasmid with CpG-free promoter).
Figure 2. DNA-induced immune response. Mice were injected i.p. with 500 μg
CpG-rich or CpG-free DNA and the levels of serum TNF-a assessed 4h postinjection.
As expected CpG-free DNA (pCpGfree-mcs and genomic calf DNA)
induced negligeable amounts of TNF-a whereas CpG-rich DNA (pDNA-CMV, CpG
ODN1826 and E. coli DNA) induced significant amounts of TNF-a. All DNAs
injected were endotoxin-free.
A B
Figure 3. LacZ expression using a pCpGfree plasmid. C57Bl6 mice were injected
with 30 μg pCpGfree-mcs (A) or pCpGfree-LacZ (B) plasmids using hydrodynamic
delivery. The livers were harvested 4 days post-inoculation and stained to determine
the expression of the CpG-free LacZ gene.
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CpG-FREE DNA 6