02 BOOK OF ABSTRACTS .indd

Optimization of electric pulse parameters for efficient
electrically-assisted gene delivery into canine skeletal
muscle
Darja Pavlin 1 , Nata{a Tozon 1 , Gregor Ser{a 2 , Azra Poga~nik 1 ,
Maja ^ema`ar 2
1
Veterinary Faculty, University of Ljubljana, Gerbi~eva 60, SI-Ljubljana; 2 Department of
Experimental Oncology, Institute of Oncology, Zalo{ka 2, SI-Ljubljana
Skeletal muscle is an attractive target tissue for delivery of therapeutic genes,
since it is usually large mass of well vascularized and easily accessible tissue with
high capacity for synthesis of proteins, which can be secreted either locally or
systemically. Electrically-assisted gene delivery into skeletal muscle of a number
of experimental animals has already been achieved using two different types of
electroporation (EP) protocols. The first one utilized only low voltage electric pulses
with long duration (e.g. 100-200 V/cm, 20-50 ms). Lately it has been shown, that
better transfection efficiency can be achieved using combination of high voltage
(HV) electric pulses (600-800 V/cm, 100 µs), which cause permeabilization of cell
membrane, followed by low voltage (LV) electric pulses to enable electrophoresis
of DNA across destabilized cell membrane.
The aim of this study was to determine optimal EP protocol for delivery of plasmid
DNA into canine skeletal muscle. For this purpose we injected 150 µg/150 µl of
plasmid, encoding green fluorescence protein (GFP), intramusculary into m.
semitendinosus of 6 beagle dogs. Electric pulses were delivered 20 minutes after
plasmid injection, with electric pulses generator Cliniporator (IGEA, Carpi, Italy),
using needle electrodes. Altogether 5 different EP protocols were utilized, each
applied to two muscles. Three of these protocols utilized combination of one
HV pulse (600 V/cm, 100 µs), followed by different number of LV pulses. Two
protocols were performed by application of LV pulses only. The control group
received only injection of plasmid without application of electric pulses. Incisional
biopsies of transfected muscles were performed 2 and 7 days after the procedure
and transfection efficiency was determined using fluorescence microscope on frozen
muscle samples.
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The highest level of GFP fluorescence in the muscle was observed in EP protocol,
using either 1 HV pulse, followed by 4 LV pulses (80 V/cm, ms, 1Hz) or with 8
LV pulses (200 V/cm, 20 ms, 1Hz). In both protocols significant GFP fluorescence
was detectable both at 2 and 7 days after transfection. Markedly lower degree of
transfection was achieved using EP protocol, utilizing 1 HV, followed by 8 LV (80
V/cm, 400 ms, 1Hz). GFP fluorescence was less pronounced compared to the first
two protocols. Furthermore, it was detectable only on muscle samples, taken at day
2 after electrotransfection. No GFP fluorescence was detectable either at 2 or 7 days
after electrotransfection on muscle samples, taken from control group and from
groups, where 1 HV, followed by 1 LV (80 V/cm, 400 ms) or 6 LV pulses (100 V/cm,
60 ms, 1Hz) were used.