Download transfer of plasmid dna and oligonucleotides into

TRANSFER OF PLASMID DNA AND OLIGONUCLEOTIDES INTO SKELETAL
MUSCLE BY MEANS OF CATIONIC LIPID-BASED VECTORS
Andrea Ditadi*, Alberto Malerba*, Gianluca Occhi*, Pier Giorgio Gamba¶, Ilaria
Scambi, Ian McLachlanª, David Baroni*, Libero Vitiello*
* Dept. of Biology, ¶ Dept. of Pediatric Surgery, University of Padova, Italy; ª Protiva
Biotherapeutics Inc., Burnaby, BC, Canada
The aim of our project is to develop a lipid-based gene delivery system capable of
introducing therapeutic DNA or antisense morpholino oligonucleotides into skeletal
muscle after systemic delivery.
We have used both DODAC-based lipopoliplexes and SPLP (stabilized plasmid-lipid
particles). We had already shown that intra-arterial delivery yielded good results in
skeletal muscle and we have optimized our surgical technique to inject the lipid-DNA
complexes in the femoral artery.
Experiments carried out on adult rats in which muscle regeneration was chemically
induced in the Tibialis Anterior muscles of the lower limb showed that SPLP containing
the luc reporter gene yielded expression levels higher than 1 ng luciferase per mg of
muscle extract, while a GFP-coding plasmid yielded approximately 10% of transfected
fibers. Importantly, we also showed that such high levels are maintained for at least three
weeks after a single administration without affecting myoblasts’ viability and
differentiation, both in vitro and in vivo.
In our latest experiments we have transferred this protocol to a murine model, using
C57BL6 animals. Somewhat surprisingly, SPLP yielded disappointing results in mice,
with transfection levels 10 to 100-fold lower than in rat. Following a series of recent
reports about exon-skipping therapeutic approaches, we have then applied our surgical
protocol to the delivery of oligonucleotide-loaded lipoplexes to murine skeletal. Our
initial results are quite encouraging, in that we have been able to restore the correct
reading frame of the dystrophin mRNA in mdx mice as old as 10 months.