A DNA controlled-release coating for gene transfer: transfection in skeletal and cardiac muscle.
In this paper we report a novel technique of DNA-polymer coating for gene transfer. A proprietary DNA polymer solution was used for thin-layer coating on a chromic gut suture as a model study. The coated sutures were characterized for physical properties such as coating thickness, mass of the DNA deposited on the suture, surface characteristics as determined by scanning electron microscopy, and in vitro DNA release characteristics under simulated physiologic conditions. The in vivo gene transfection using DNA-coated sutures was demonstrated in rat skeletal muscle and in canine atrial myocardium. A heat-stable human placental alkaline phosphatase (AP) plasmid was used as a marker gene. Incisions of 1 to 1.5 cm were made in the rat skeletal muscles or the canine atrial myocardium. The sites were closed with either the DNA-coated sutures or control sutures. Two weeks after the surgery, the tissue samples adjacent to the suture lines were retrieved and analyzed for AP activity. The DNA-coated sutures demonstrated a sustained release of the DNA under in vitro conditions, with an approximately 84% cumulative DNA release occurring in 26 days. An agarose gel electrophoresis of the DNA samples released from the suture demonstrated two bands, with the lower band corresponding to the input DNA (supercoiled). It seems that there was a partial transformation of the DNA from a supercoiled to an open circular form due to the polymer coating. The tissue sites, which received the DNA-coated sutures, demonstrated a significantly higher AP activity compared with the tissue sites that received control sutures. In the rat studies, the mean AP activity (square root of cpm/microgram protein) was 43.6 +/- 3.3 vs 20.6 +/- 2.1 (p = 0.001) at the control sites. Similarly, in the canine studies, the AP activity was 73.6 +/- 7.4 Vs 21.6 +/- 1.4 (p = 0.0009) at the control sites. Thus, our studies demonstrated a successful gene transfer using our DNA-polymer coating technique. This technique could be useful for coating sutures used in vascular and general surgery, and also for coating medical devices, such as stents, catheters, or orthopedic devices, to achieve a site-specific gene delivery.
Labhasetwar
V
V
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA. Pediatric Research Center, Room 1170B.
Bonadio
J
J
Goldstein
S
S
Chen
W
W
Levy
R J
RJ
D000818:Animals; D004247:DNA / Q000008:administration & dosage* / Q000737:chemistry; D003692:Delayed-Action Preparations; D004285:Dogs; D004337:Drug Carriers; D016503:Drug Delivery Systems; D015316:Genetic Therapy; D006321:Heart / Q000502:physiology*; D006801:Humans; D008297:Male; D018482:Muscle, Skeletal / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
D003692:Delayed-Action Preparations; D004337:Drug Carriers; D004247:DNA