Gene transfer into adult rat spinal cord using naked plasmid DNA and ultrasound microbubbles.
BACKGROUND
Although gene therapy might become a promising approach to treat spinal cord injury, the safety issue is a serious consideration in human gene therapy. Plasmid DNA transfer is safer than viral vectors, but the transfection efficiency is quite low. To overcome the problem, we applied the ultrasound microbubbles-mediated transfection method to the spinal cord in adult rats, since ultrasound microbubbles have been reported to be efficient to increase transfection efficiency in various tissues.


METHODS
After exposing T9-10 spinal cord with a laminectomy, we injected a mixture of naked plasmid DNA and microbubbles into cerebrospinal fluid by lumbar puncture. Then, the T9-10 spinal cord was exposed to ultrasound.


CONCLUSIONS
An ultrasound intensity of 0.4-0.5 W/cm2 significantly increased luciferase expression up to approximately 15-60-fold at the insonated level as compared to naked plasmid DNA alone. Luciferase activity could be detected at least up to 7 days after transfection, while the expression level was almost returned to undetectable level at 14 days after transfection. The transfected cells were mainly meningeal cells in the surface of insonated spinal cord. There was no obvious evidence of worsening of neurological deficits as compared to rats transfected with naked plasmid DNA alone or untransfected rats. Similarly, successful gene transfer was also achieved in the insonated T9-10 spinal cord after spinal cord injury. Overall, the present study demonstrated the feasibility of ultrasound microbubbles-mediated plasmid DNA transfer into the target level of the spinal cord.
Division of Clinical Gene Therapy, Graduate School of Medicine, Osaka University, Yamada-oka 2-2, Suita, Osaka 565-0871, Japan.
Munehisa
M
Shimamura
Naoyuki
N
Sato
Yoshiaki
Y
Taniyama
Hitomi
H
Kurinami
Hiroyuki
H
Tanaka
Toshihiro
T
Takami
Toshio
T
Ogihara
Masaya
M
Tohyama
Yasufumi
Y
Kaneda
Ryuichi
R
Morishita
D000818:Animals; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015870:Gene Expression; D015316:Genetic Therapy; D006801:Humans; D008297:Male; D045423:Microbubbles; D009043:Motor Activity; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013116:Spinal Cord / Q000033:anatomy & histology / Q000473:pathology / Q000502:physiology*; D014162:Transfection; D014465:Ultrasonics
D004247:DNA