Volume 2, Issue 3, Pages 204-210 (September 2000) High-Level Protein Secretion into Blood Circulation after Electric Pulse-Mediated Gene Transfer into Skeletal Muscle  Mickaël Bettan, Florence Emmanuel, Raphaël Darteil, Jean-Michel Caillaud, Fabienne Soubrier, Pia Delaere, Didier Branelec, Abderrahim Mahfoudi, Nicolas Duverger, Daniel Scherman  Molecular Therapy  Volume 2, Issue 3, Pages 204-210 (September 2000) DOI: 10.1006/mthe.2000.0117 Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 1 hSeAP concentration in plasma after im injection of increasing amounts of pCMV-hSeAP. Groups of 6-week-old female C57Bl/6 mice were injected unilaterally (except for 400 μg) into tibialis cranialis muscle, in the absence of electric pulses (open circles) or with subsequent electrotransfer (closed circles), with 0.1 μg (n = 5), 0.3 μg (n = 5), 1 μg (n = 5), 10 μg (n = 10), or 400 μg (200 μg in both tibialis cranialis muscles, n = 15) of pCMV-hSeAP in 30 μl of 150 mM NaCl. in each injected muscle. Seven days after injection, blood samples were collected in heparinized tubes and human placental alkaline phosphatase assayed in duplicate in plasma using Phospha-Light kit (Tropix). Values are means ± SEM. Molecular Therapy 2000 2, 204-210DOI: (10.1006/mthe.2000.0117) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 2 Time course of hSeAP in plasma of immunocompetent mice after im injection of pCMV-hSeAP. Six-week-old female C57Bl/6 mice were injected bilaterally into the tibialis cranialis muscle with 0 μg (n = 5, open triangles), 15 μg (n = 10, open circles), or 15 μg followed by electric pulses (n = 10, closed circles) of pCMV-hSeAP plasmid in 30 μl of 150 mM NaCl in each injected muscle. At the indicated time after injection, blood samples were collected and alkaline phosphatase assay was performed. Values are means ± SEM. Molecular Therapy 2000 2, 204-210DOI: (10.1006/mthe.2000.0117) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 3 Time course of hSeAP in plasma of SCID mice after im injection of pCMV-hSeAP. Six-week-old female SCID mice were injected bilaterally into the tibialis cranialis muscle with pCMV-hSeAP plasmid in 30 μl of 150 mM NaCl in each injected muscle: 0 μg (open triangles), 15 μg (open circles), 0 μg followed by electric pulse (closed triangles), or 15 μg followed by electric pulses (closed circles). At the indicated time after injection, blood samples were collected and alkaline phosphatase assay performed. Values are means ± SEM (n = 10). Molecular Therapy 2000 2, 204-210DOI: (10.1006/mthe.2000.0117) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 4 Time course of the concentration of human FIX in plasma of immunocompetent and immunodeficient mice and of anti-hFIX antibody in plasma of immunocompetent mice after a single im injection of pCMV-hFIX plasmid. Six-week-old female C57Bl/6 mice (closed squares), SCID mice (closed circles), and Nude mice (closed triangles) were injected bilaterally into the tibialis cranialis muscle with 15 μg pCMV-hfactor IX plasmid in 30 μl 150 mM NaCl for each leg and electrotransferred. At the indicated time after injection, blood samples were collected. Human Factor IX and anti-human Factor IX (open circles) were assayed in plasma. Values are means ± SEM. Molecular Therapy 2000 2, 204-210DOI: (10.1006/mthe.2000.0117) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 5 Factor IX expression in mouse tibialis cranialis muscle transversal section. Six-week-old female Nude mice were injected into the tibialis cranialis muscle with 15 μg pCMV-hfactor IX plasmid, in 30 μl 150 mM NaCl and electrotransferred. Three days after injection, human Factor IX expression was detected by immunohistochemistry. Electrotransferred muscles were analyzed by a classic streptavidin–biotin immunohistological assay. The procedure was done using a Ventana medical system. The specific antibody was a polyclonal rabbit anti-human FIX antibody (Sigma Immunohistochemicals). Molecular Therapy 2000 2, 204-210DOI: (10.1006/mthe.2000.0117) Copyright © 2000 American Society for Gene Therapy Terms and Conditions