Transcutaneous Ultrasound Augments Naked DNA Transfection of Skeletal Muscle

Abstract

This study was designed to test the hypothesis that transcutaneous ultrasound (US) exposure may augment the transfection efficiency and biological outcome associated with nonviral DNA gene transfer. Hindlimb muscles of New Zealand White rabbits were transfected with the reporter plasmid pCMV-beta, with or without US exposure. Optimization studies employed US exposure at various frequencies, mechanical indices, duty cycles, durations of exposure, and exposure time points. Based on these results, we explored the effect of US exposure on nonviral gene transfer of vascular endothelial growth factor (VEGF, phVEGF165) to promote neovascularization of ischemic hindlimbs. Ultrasound at 1 MHz, 100 W/cm(2), 6% duty cycle, and 5 minutes exposure time, applied immediately following DNA injection, was found to be the most effective among the settings tested, increasing beta-galactosidase expression approximately 20 fold. Compared with US exposure alone, or phVEGF165 only, phVEGF165 + US exposure yielded a statistically significant improvement in revascularization, as determined by calf blood pressure ratio, angiographic score, intravascular Doppler blood flow, and capillary/myocyte ratio. These data demonstrate that ultrasound, when applied directly after intramuscular gene transfer, significantly increases transfection efficiency in vivo. The biological significance of this finding was confirmed by augmented limb perfusion in response to US exposure and naked VEGF DNA.

Transcription factor decoy oligonucleotide-based therapeutic strategy for renal disease

“…40 Most sonoporation studies have been carried out on cultured cells, [39][40][41][42][43][44] tumors in vivo, [45][46][47][48] or skeletal muscle. [49][50][51] Moreover, we have recently found that this approach is applicable to gene transfer into the artery. 52 However, to our knowledge, there has been no previous investigation of whether ultrasound is able to enhance plasmid DNA transfer into the kidney.…”

Ultrasound-mediated gene transfection: problems to be solved and future possibilities

“…The combination of USMT and the VEGF gene improved angiogenesis significantly more than injection of the VEGF gene alone. 14 …”

“…To date, enhanced USMT efficiency achieved by using microbubbles has been reported in such organs as the uterus, 25 the endovascular organs, 26 the skeletal muscles, [27][28][29] and the kidneys 30,31 (Table 2). In no case did 13 2 MHz 12-fold In vivo: rabbit model of arterial mechanical overdilation injury 50 W/cm 2 16 min Manome et al 11 1 MHz 2.0 ± 0.63 β-galactosidase activity In vivo: mouse s.c. model MC38 murine colon 20 W/cm 2 adenocarcinoma cells 30 s (focusing) Lawrie et al 10 1 MHz 3.0 ± 2.0 LU/ng protein (7.5-fold) In vitro: vascular smooth muscle cells 0.4 W/cm 2 30 min Schratzberger et al 14 1 MHz 100 W/cm 2 129.9 ± 16.8 RLU/mg protein (20.4-fold) In vivo: rabbit hind limb muscle 5 min s.c., Subcutaneously transplanted tumor model 28 Albumin bubbles 20 kHz Data not shown In vivo: porcine coronary artery Endoh et al 25 Optison (50%) 1 MHz 3 × 10 6 RLU/mg In vivo: pregnant mice, intrauterine −0.6 MI protein (−10 3 -fold) Frenkel et al 24 Albumin bubbles 1.3 MHz 41 ± 3% In vitro: 293 cell line 1.6 MI Taniyama et al 26 Optison (25%) 1 MHz >1000-fold In vivo: rat carotid artery 2.5 W/cm 2 Taniyama et al 27 Optison (75%) 1 MHz 10-fold In vivo: rat ischemic muscle 2.5 W/cm 2 Azuma et al 31 Optison 2 MHz 3691 ± 658 In vivo: rat renal allograft model 2.5 W/cm 2 LU/kidney Lu et al 29 Optison 1 MHz 12-fold In vivo: mouse skeletal muscle 3 W/cm 2 Nakashima et al 33 Optison (5%-10%) 1 MHz 50% In vivo: dental pulp stem cells 0.5 W/cm 2 Nozaki et al 42 Levovist (10 mg/ml) 1 MHz 0.89%-15.3% In vitro: prostate cancer cell 3.6 W/cm 2 USMT produce the obvious tissue damage typically observed on application of other physical transfection modalities. 32,33 This modality has attracted attention as a minimally invasive method of transfection and has begun to see use in the field of regenerative medicine, where avoiding tissue damage is of primary importance.…”

“…Because cavitation bubbles are rarely generated at higher frequencies, however, relatively higher ultrasound intensity (20-100 W/cm 2 ) or longer sonication time (3-30 min) was required, [10][11][12][13][14] leading to concerns about targettissue injury (Table 1). Moreover, USMT has not produced satisfactory transfection efficiency.…”

Nucleic Acid Delivery and Localizing Delivery with Magnetic Nanoparticles

“…Interestingly, ultrasound alone has been shown to enhance gene delivery to cell lines [233,234], skeletal muscle [235], and tumors [236]. Associating nucleic acids with microbubbles, applying such compositions in vitro and in vivo under exposure of the target tissue to ultrasound is a highly effective method for triggering localized delivery of nucleic acids and drugs in general in a variety of tissues [142,226,[237][238][239][240][241][242][243][244][245][246].…”