Delivery of Na/I symporter gene into skeletal muscle using nanobubbles and ultrasound: visualization of gene expression by PET.
The development of nonviral gene delivery systems is essential in gene therapy, and the use of a minimally invasive imaging methodology can provide important clinical endpoints. In the current study, we present a new methodology for gene therapy-a delivery system using nanobubbles and ultrasound as a nonviral gene delivery method. We assessed whether the gene transfer allowed by this methodology was detectable by PET and bioluminescence imaging.


METHODS
Two kinds of reported vectors (luciferase and human Na/I symporter [hNIS]) were transfected or cotransfected into the skeletal muscles of normal mice (BALB/c) using the ultrasound-nanobubbles method. The kinetics of luciferase gene expression were analyzed in vivo using bioluminescence imaging. At the peak of gene transfer, PET of hNIS expression was performed using our recently developed PET scanner, after (124)I injection. The imaging data were confirmed using reverse-transcriptase polymerase chain reaction amplification, biodistribution, and a blocking study. The imaging potential of the 2 methodologies was evaluated in 2 mouse models of human pathology (McH/lpr-RA1 mice showing vascular disease and C57BL/10-mdx Jic mice showing muscular dystrophy).


RESULTS
Peak luciferase gene activity was observed in the skeletal muscle 4 d after transfection. On day 2 after hNIS and luciferase cotransfection, the expression of these genes was confirmed by reverse-transcriptase polymerase chain reaction on a muscle biopsy. PET of the hNIS gene, biodistribution, the blocking study, and autoradiography were performed on day 4 after transfection, and it was indicated that hNIS expression was restricted to the site of plasmid administration (skeletal muscle). Similar localized PET and (124)I accumulation were successfully obtained in the disease-model mice.


CONCLUSIONS
The hNIS gene was delivered into the skeletal muscle of healthy and disease-model mice by the ultrasound-nanobubbles method, and gene expression was successfully visualized with PET. The combination of ultrasound-nanobubble gene transfer and PET may be applied to gene therapy clinical protocols.
Watanabe
Yukiko
Y
Graduate School of Biomedical Engineering, Tohoku University, Aoba-ku, Sendai, Japan.
Horie
Sachiko
S
Funaki
Yoshihito
Y
Kikuchi
Youhei
Y
Yamazaki
Hiromichi
H
Ishii
Keizo
K
Mori
Shiro
S
Vassaux
Georges
G
Kodama
Tetsuya
T
D000818:Animals; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006801:Humans; D007457:Iodine Radioisotopes / Q000493:pharmacokinetics; D007700:Kinetics; D008156:Luciferases / Q000235:genetics; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism*; D049329:Nanostructures; D049268:Positron-Emission Tomography; D027981:Symporters / Q000235:genetics*; D014465:Ultrasonics; D000099209:Sodium-Iodide Symporters
D007457:Iodine Radioisotopes; D008156:Luciferases; D027981:Symporters; D000099209:Sodium-Iodide Symporters