1. Volume 17, Issue 9, Pages 1637-1642 (September 2009)
Optimization of Skin Electroporation in Mice to Increase Tolerability of DNA Vaccine Delivery to Patients 
Anna-Karin Roos, Fredrik Eriksson, Derin C Walters, Pavel Pisa, Alan D King 
Molecular Therapy 
Volume 17, Issue 9, Pages (September 2009)
DOI: /mt
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
PSA-specific CD8+ T-cell responses in mice after vaccination using electroporation protocols with different pulse intervals. C57Bl/6 mice were immunized intradermally once with 20 µg pVax-PSA plasmid in 20-µl phosphate-buffered saline on each flank and electroporation was applied. Splenocytes were isolated on day 14 after immunization and cells were stimulated for 5 hours with 100 nmol/l psa65–73 or control peptide GP33. Activated CD8+ T cells were quantified using intracellular cytokine staining for IFNγ and analyzed by flow cytometry. (a) Schematic overview of electroporation protocols used in this study showing pulse (P) and interval (I) duration. (b) PSA-specific IFNγ-producing CD8+ T-cell levels induced after intradermal delivery of pVax-PSA and application of the indicated electroporation protocol. The experiment was performed twice with four mice per group and pooled data is shown. Background levels after GP33 stimulation were 0–0.15%. Bars represent mean ± SD (n = 8). Ctrl, control; PSA, prostate-specific antigen.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Luciferase gene expression in skin after DNA delivery using PA slow and PA fast electroporation protocols. Balb/c mice were injected intradermally with 10 µg of pVax-luc plasmid in 20-µl PBS on each flank and were subjected to either PA slow or PA fast electroporation protocols. At day 2, 8, 30, and 62 after DNA vaccination, mice were injected intraperitoneally with 100-µl/10-g mouse body weight with 15 mg/ml of D-luciferin substrate solution. Twenty minutes after substrate injection, the in vivo luciferase expression was visualized using the IVIS 100 in vivo imaging system. (a) Representative bioluminescent image showing luciferase expression in skin 48 hours after DNA delivery. The scale shows intensity of luminescence (photons/s/cm2). The electroporation protocols used for individual mice are indicated. (b) Time course of in vivo luciferase expression after DNA delivery with PA slow (filled squares) and PA fast (filled triangles) protocols. The experiment was performed twice with 2–3 mice per group (two luciferase injection sites per mouse) and one representative experiment is shown. Error bars represent (±) SD (n = 4). PA, PulseAgile; PBS, phosphate-buffered saline; ROI, region of interest.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
IFNγ production and degranulation by PSA-specific CD8+ T cells after PSA DNA vaccination using PA fast protocol with and without application of anesthetic cream. C57Bl/6 mice were immunized intradermally once with 20-µg pVax-PSA plasmid in 20-µl phosphate-buffered saline on each flank and electroporation was applied. About 45–55 minutes prior to plasmid injection and electroporation, emla cream was administered topically at the injection area to one group of mice. Splenocytes were isolated on day 14 after immunization and cells were stimulated for 5 hours with 100 nmol/l psa65–73 or control peptide GP33. Activated CD8+ T cells were quantified using intracellular cytokine staining for IFNγ and surface staining for CD107a and analyzed by flow cytometry. (a) Levels of PSA-specific IFNγ production by CD8+ T cells by individual mice after intradermal delivery of pVax-PSA using PA fast protocol with and without topical addition of emla cream. Shown is specific response to psa65–73 (filled bars) and background response to GP33 (open bar). (b) Levels of PSA-specific CD8+ T cells expressing CD107a in individual mice after intradermal delivery of pVax-PSA using PA fast protocol with and without topical application of emla cream. Shown is specific response to psa65–73 (filled bars) and background response to GP33 (open bars). Pooled data from two individual experiments is shown. emla, eutectic mixture of lidocaine and prilocaine; PA, PulseAgile; PSA, prostate-specific antigen.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Cytotoxicity assay of in vitro restimulated PSA-specific CD8+ T cells after PSA DNA vaccination using PA fast protocol with and without application of anesthetic cream. Mice were immunized intradermally once with 20-µg pVax-PSA plasmid in 20-µl phosphate-buffered saline on each flank and electroporation was applied. About 45–55 minutes prior to plasmid injection and electroporation, emla cream was administered topically at the injection area to one group of mice. Splenocytes were collected 14 days after immunization and restimulated in vitro for 5 days with the psa65–73 peptide. The splenocytes were then tested for cytolytic activity against PSA expressing (filled bars) and control target cells (open bars). Shown is the lysis at 30:1 effector target (e:t) ratio. The mean of two different experiments is shown. Error bars represent SD (n = 10). emla, eutectic mixture of lidocaine and prilocaine; PA, PulseAgile; PSA, prostate-specific antigen.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions