gene expression and immune response kinetics using electroporation-mediated dna delivery to muscle...

Gene expression and immune response kineticsusing 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, Norway2Department of Orthopaedics, Lund University Hospital, Lund, Sweden

3Inovio AS, Box 180 Vindern, 0319 Oslo, Norway

THE JOURNAL OF GENE MEDICINEJ Gene Med 2005; 7: 218–227.

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Mammalianexpression

vector

Method overview

mouse muscle

coding for protein X

X

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

injection only

Luciferase activity in muscle extracts after 5 days

(n=10, 5, 10, 2)

Amount of secreted Alkaline Phosphatase in blood (n=6)unipolar

bipolar

Unipolar provides a better transfection and a higher blood concentration.

Fig1

Level drops faster in blood than in muscle, indicatinginactivation or removal in theblood in addition to reducedexpression in the muscle

A-D: unipolar, 200VE: bipolar, 200V

A: day 7 (high transfection, alsohigh number of necrotic fibers)

B: day14

C: day 56

D: Quantification of positivefibers 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.

Fig3

Fig4

Unipolar pulse damages the muscle fibers to a greater extend

CPK (released from ruptured fibers) in blood(n=5 except for groups 5 and 6 where n=4)

histological evaluation(n=10, 8)

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 andeffectiveness of transfection/electroporation

Fig6

IgG1 levels correlates with a high differenceof expression level between day 7 and day14

X

Y= Z

If Y >> X, Z is very small.

IgG1 and 2 levels correlates with a high differenceof expression level between day 7 and day14

Highlight the central role of IgG1in early reduction of SEAP blood levels

IgG1and 2 are both involved in the latereduction of SEAP blood levels

i.e.: direct & cellular immune response

Fig7Bipolar voltage seems more effective on the long run

Conclusions:

- unipolar pulse are more effective at tranfecting the plasmid, but induce more damagewhich could influence the immune response.

- bipolar pulses are less effective at transfecting the plasmid but generally induce a longerlasting 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 cellsand the expansion of the mesangial ECM.

Plasma lvl at day4244.4±89.8 ng/mL

(if molecule is 100kDA,then = 2.4nM)

other results