Gene expression and immune response kinetics using electroporation-mediated DNA delivery to muscle Eirik Grønevik1*, Fredik Vult von Steyern2, John Magne Kalhovde1 Torunn Elisabeth Tjelle3, Iacob Mathiesen3 1Department of Physiology, University of Oslo, Box 1103 Blindern, 0316 Oslo, Norway 2Department of Orthopaedics, Lund University Hospital, Lund, Sweden 3Inovio AS, Box 180 Vindern, 0319 Oslo, Norway THE JOURNAL OF GENE MEDICINE J Gene Med 2005; 7: 218–227.
- + Method overview coding for protein X Mammalian expression vector X mouse muscle
SEAP Phospha-Light™ Secreted Alkaline Phosphatase Reporter Gene Assay System SEAP gene product is a truncated form of human placental alkaline phosphatase alkaline phosphatase approx 140kDa = 140kg/mole =140ng/pmol (MW of hydrogen is 1g/mol=1Da) 400ng/mL = 2.8pmol/mL = 2.8nM
Fig1 bipolar unipolar Amount of secreted Alkaline Phosphatase in blood (n=6) Luciferase activity in muscle extracts after 5 days (n=10, 5, 10, 2) Unipolar provides a better transfection and a higher blood concentration.
Level drops faster in blood than in muscle, indicating inactivation or removal in the blood in addition to reduced expression in the muscle
Fig3 A-D: unipolar, 200V E: bipolar, 200V A: day 7 (high transfection, also high number of necrotic fibers) B: day14 C: day 56 D: Quantification of positive fibers at Day 7 and Day 14 (n=5) E: day7, less necrosis, but also invasion of mononuclear cells. Rapid disparition of transfected fibers, antigenicity of the protein transfected is capital here.
Fig4 CPK (released from ruptured fibers) in blood (n=5 except for groups 5 and 6 where n=4) histological evaluation (n=10, 8) Unipolar pulse damages the muscle fibers to a greater extend
Fig5 IgG1 is comes in early, IgG2 comes late, possibly more involved with the cellular aspect of the immune response The levels of expression of both antibodies are related to the intensity and effectiveness of transfection/electroporation
Fig6 X = Z Y If Y >> X, Z is very small. IgG1 levels correlates with a high difference of expression level between day 7 and day14 Highlight the central role of IgG1 in early reduction of SEAP blood levels IgG1 and 2 levels correlates with a high difference of expression level between day 7 and day14 IgG1and 2 are both involved in the late reduction of SEAP blood levels i.e.: direct & cellular immune response
Fig7 Bipolar voltage seems more effective on the long run
Conclusions: - unipolar pulse are more effective at tranfecting the plasmid, but induce more damage which could influence the immune response. - bipolar pulses are less effective at transfecting the plasmid but generally induce a longer lasting expression, as seen in blood levels. - allogenic protein can be expressed a ng/mL level up to 58 days, syngenic protein can last for more that 80 days.
Functional example: • Kidney Int. 2001 Jun;59(6):2134-45 • Electroporation-mediated PDGF receptor-IgG chimera • gene transfer ameliorates experimental glomerulonephritis.Nakamura H, Isaka Y, Tsujie M, Akagi Y, Sudo T, Ohno N, Imai E, Hori M. • Department of Internal Medicine and Therapeutics, • Osaka University Graduate School of Medicine, Suita, Japan. • glomerulonephritis is characterized by the proliferationj of glomerular mesangial cells • and the expansion of the mesangial ECM.
Plasma lvl at day4 244.4±89.8 ng/mL (if molecule is 100kDA, then = 2.4nM) other results