1. CpG Methylation of a Plasmid Vector Results in Extended Transgene Product Expression by Circumventing Induction of Immune Responses 
A. Reyes-Sandoval, H.C.J. Ertl 
Molecular Therapy 
Volume 9, Issue 2, Pages (February 2004)
DOI: /j.ymthe
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
HpaII digestion of the methylated pSG5rab.gp vector. A total of 1 μg of unmethylated and methylated pSG5rab.gp was incubated with HpaII restriction enzyme at 37°C. After 24 h, 100 ng of DNA was separated in a 1% agarose gel. Lane 1, untreated unmethylated pSG5rab.gp vector. Lane 2, untreated CpG-methylated pSG5rab.gp vector. Lane 3, HpaII-digested unmethylated pSG5rab.gp vector. Lane 4, HpaII-digested CpG-methylated pSG5rab.gp vector. Lane 5, 100 bp molecular weight marker.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
In vivo expression of rabies glycoprotein gene. Groups of five C3H/He mice were injected with 150 μg of CpG-methylated or unmethylated pSG5rab.gp vector into each of the lower leg muscles. Muscles were recovered 3 days later, and RNA was isolated, treated with DNase, reverse transcribed, and amplified by a real-time RT-PCR using rabies virus glycoprotein gene-specific primers. Data indicate number of RNA copies per 1 × 106 molecules of GAPDH. The difference between the two groups was statistically not significant (P > 0.8 by Student t test). Muscle tissues from naı¨ve mice were used as a control; a cDNA sequence from these samples was amplified only with the GAPDH and not with the rabies virus glycoprotein gene primers (not shown).
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
Longevity of transgene product expression in muscles of mice inoculated with unmethylated or CpG-methylated pSG5rab.gp vector. Groups of five C3H/He mice were injected with 150 μg of methylated and unmethylated pSG5rab.gp vectors into each of the lower leg muscles. RNA was isolated after 4 and 8 weeks, reverse transcribed, and tested for presence of rabies virus glycoprotein-encoding sequences by a nested PCR using samples from individual muscles. PCR products were separated in 1% agarose gels together with a molecular weight (MW) standard. Bands for the housekeeping gene are shown on the top. Amplification of the rabies virus glycoprotein gene by a single and by a nested PCR is shown in the middle and bottom rows, respectively.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

5. Fig. 4
Quantification of DNA and RNA by real-time PCR and RT-PCR. DNA and RNA were isolated from individual lymph nodes 3 days after inoculation with 150 μg of unmethylated or methylated pSG5rab.gp vector. Lymph nodes from naı¨ve mice were used as controls. (A) Quantification of rab.gp DNA in lymph nodes by PCR. Calculated P values are unmethylated/methylated pSG5rab.gp, P < 0.05; unmethylated or methylated pSG5rab.gp/pSG5, P < The data indicate number of DNA copies per 1 × 109 molecules of housekeeping gene GAPDH. (B) Quantitative expression of rab.gp transcripts in lymph nodes. Unmethylated/methylated pSG5rab.gp, P = 0.45; unmethylated or methylated pSG5rab.gp/pSG5, P < (C) Quantitative expression of IFN-γ transcripts in lymph nodes. Unmethylated/methylated pSG5rab.gp, P < 0.005; unmethylated or methylated pSG5rab.gp/pSG5, P < (D) Quantitative expression of CD86 transcripts in lymph nodes. Unmethylated/methylated pSG5rab.gp or pSG5, P < ; methylated pSG5rab.gp/pSG5, P = 0.4. The data indicate number of transcripts per 1 × 106 molecules of housekeeping gene GAPDH for (B), (C), and (D). Statistical analysis was performed using a Student t test.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

6. Fig. 5
Antibody response to unmethylated and methylated pSG5rab.gp vector prior and subsequent to a booster immunization with the unmethylated pSG5rab.gp vector. (A) Groups of five C3H/He mice were inoculated intramuscularly with 10 μg of unmethylated pSG5rab.gp (open squares), 10 μg of methylated pSG5rab.gp (closed squares), and 10 μg of empty pSG5 (squares with cross) vector. Sera were harvested 4 and 8 weeks later and tested for antibodies by an ELISA. Unmethylated/methylated pSG5rab.gp or pSG5, P < (B) The same groups of mice were boosted with 10 μg of the same vector used for the priming. Mice were bled 4 and 8 weeks later and screened for antibodies by an ELISA. (C) Groups of six mice were injected with 300 μg of unmethylated and methylated vector expressing the rabies glycoprotein. Mice were bled 4 and 8 weeks after priming and sera were screened for antibodies using an ELISA. Unmethylated/methylated pSG5rab.gp, P < 0.001; unmethylated/pSG5 vector, P < 0.001; methylated/pSG5 vector, P < (D) The same groups of mice (B) were boosted with 50 μg of unmethylated pSG5rab.gp vector. An additional group of naı¨ve C3H/He mice was inoculated with 50 μg of pSG5 vector each (X). All groups were bled 4 and 8 weeks later and screened for antibodies using an ELISA. Methylated pSG5rab.gp/pSG5, P < 0.002; unmethylated pSG5rab.gp, methylated pSG5rab.gp, pSG5/control, P < Data show means ± SD for triplicate samples. Statistical analysis was performed with a Student t test for the first two dilutions for the sera harvested 8 weeks after immunization.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

7. Fig. 6
The antibody isotype in mice pretreated with the methylated pSG5rab.gp vector. Mice were injected first with 10 μg of unmethylated or methylated pSG5rab.gp vector or pSG5 vector and subsequently boosted with 50 μg of unmethylated pSG5rab.gp vector (see also Fig. 5D). Sera harvested 8 weeks after immunization with 50 μg of unmethylated pSG5rab.gp vector and sera from naı¨ve mice (controls) were tested for IgG1 (striped bars) and IgG2a (black bars) antibodies to rabies virus. Data were obtained with a 1:400 dilution of the sera and are means ± SD of duplicate values.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

8. Fig. 7
Lymphokine release by antigen-pulsed splenocytes harvested from mice pretreated with the CpG-methylated pSG5rab.gp vector. Mice from groups similar to those described in the legends to Figs. 3–5 were euthanized (A, C) 8 weeks after treatment with methylated or unmethylated pSG5rab.gp or pSG5 vector or (Control, B, D) 8 weeks after the follow-up immunization with 50 μg of the unmethylated pSG5rab.gp vector or the pSG5 vector and tested for cytokine secretion. (A) Splenocytes from mice injected with 10 μg of unmethylated pSG5rab.gp, methylated pSG5rab.gp, or pSG5 vector were harvested 8 weeks after the second immunization and stimulated for 48 h with ERA-BPL virus (black bars) or no antigen (striped bars). Supernatants were tested for induction of proliferation of HT-2 cells. Data show the mean [3H]TdR incorporation of triplicate samples in cpm ± SD. Unmethylated pSG5rab.gp/methylated pSG5rab.gp or pSG5, P < (B) The same supernatants described in (A) were tested for IFN-γ with an ELISA against a cytokine standard. Data show the mean units of IFN-γ ± SD from triplicate samples. Unmethylated pSG5rab.gp/methylated pSG5rab.gp, P < (C) Mice treated as described in (A) were inoculated 8 weeks after the second inoculation with 50 μg of pSG5rab.gp vector or pSG5 vector (Control). Splenocytes were harvested 8 weeks later and cultured with ERA-BPL virus (black bars) or no antigen (striped bars). Supernatants were harvested 48 h later and tested for IL-2 with an ELISA against an IL-2 standard. Data show the mean IL-2 in pg/ml ± SD of triplicate samples. Methylated pSG5rab.gp/unmethylated pSG5rab.gp, P < 0.05; methylated pSG5rab.gp/pSG5, P < (D) The same supernatants described in (C) were tested for IFN-γ using an ELISA as described for (B). Methylated pSG5rab.gp/unmethylated pSG5rab.gp, P = 0.02; methylated pSG5rab.gp/pSG5, P = 0.05.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

9. Fig. 8
Effects of pretreatment of mice with the CpG-methylated pSG5rab.gp vector on the rabies virus-specific antibody response elicited by the AdH5rab.gp construct. Groups of five C3H/He mice were inoculated 2× with 10 μg of unmethylated pSG5rab.gp (open squares), methylated pSG5rab.gp (closed squares), and pSG5 vector (squares with cross). Eight weeks after the second inoculation, mice were injected intramuscularly with (A) 1 × 104 or (B) 5 × 104 pfu of AdHu5rab.gp virus. Sera were harvested 5, 10, and 24 days later and screened for antibodies to the rabies glycoprotein using an ELISA. Mice inoculated with the pSG5 vector once 8 weeks before harvesting of the sera were used as a negative control (X). Data show the means ± SD of triplicate values.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions