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Gene expression and immune response kinetics using electroporation-mediated DNA delivery to muscle Eirik Grønevik 1 *, Fredik Vult von Steyern 2 , John Magne Kalhovde 1 Torunn Elisabeth Tjelle 3 , Iacob Mathiesen 3

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Presentation Transcript
slide1

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.

slide2

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+

Method overview

coding for protein X

Mammalian

expression

vector

X

mouse muscle

slide3

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

slide5

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.

slide6

Level drops faster in blood

than in muscle, indicating

inactivation or removal in the

blood in addition to reduced

expression in the muscle

slide7

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.

slide8

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

slide9

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

slide10

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

slide11

Fig7

Bipolar voltage seems more effective on the long run

slide12

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.

slide13

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.
slide14

Plasma lvl at day4

244.4±89.8 ng/mL

(if molecule is 100kDA,

then = 2.4nM)

other results