Microbubble-enhanced ultrasound for gene transfer into living skin equivalents.
BACKGROUND
Gene transfer to skin is an attractive therapeutic approach because of the accessibility of the skin and the high rate of cure for many cutaneous diseases. However, safety concerns over viral vectors and the low efficiency of most non-viral gene transfer techniques have encumbered their clinical application for gene transfer. By contrast, efficient gene transfers into various cell types using microbubble-enhanced ultrasound has been reported.


OBJECTIVE
The purpose of this study was to investigate whether ultrasound with microbubble enhancement allowed effective transfer of foreign genes into living skin equivalents (LSEs).


METHODS
Microbubbles and plasmid DNA encoding green fluorescent protein (GFP) were added to the dermal-epidermal junctions of LSEs, which were then exposed to ultrasound. The LSEs were harvested at different time points to investigate transgene expression using confocal laser microscopy. Transfected LSEs were also transplanted onto nude mice, and the in vivo transgene expression was observed.


RESULTS
From days 2 to 7 after transfection, most GFP-positive cells continued to migrate upward from the basal layer, while other GFP-positive cells lagged behind or remained in the basal layer on days 5 and 7. Transfection resulted in 20-30% GFP-positive cells. Multiple transfections further increased the percentage of transfected cells and resulted in multi-layer transgene expression. Grafts from the transfected LSEs survived on nude mice and continued to express GFP up to 2 weeks post-transplantation.


CONCLUSIONS
Gene transfer into LSE using ultrasound with microbubble enhancement is an effective alternative to viral and non-viral methods.
Yang
Lujun
L
Department of Dermatology, Ehime University School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan.
Shirakata
Yuji
Y
Tamai
Katsuto
K
Dai
Xiuju
X
Hanakawa
Yasushi
Y
Tokumaru
Sho
S
Yahata
Yoko
Y
Tohyama
Mikiko
M
Shiraishi
Ken
K
Nagai
Hiroshi
H
Wang
Xiaoling
X
Murakami
Shinji
S
Sayama
Koji
K
Kaneda
Yasufumi
Y
Hashimoto
Koji
K
D000818:Animals; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics*; D051379:Mice; D008819:Mice, Nude; D045423:Microbubbles; D018613:Microscopy, Confocal; D010957:Plasmids; D012867:Skin / Q000000981:diagnostic imaging*; D012871:Skin Diseases / Q000628:therapy; D014162:Transfection / Q000379:methods*; D019076:Transgenes; D014463:Ultrasonography
D049452:Green Fluorescent Proteins