Sonic Hedgehog cDNA-plasmid Vector Transduction by Microbubble-Enhanced Sonoporation for Ectopic Bone Formation

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We investigated ectopic bone formation in mouse femoral muscles by direct gene transfer of Sonic hedgehog (SHH) cDNA encoding amino-terminal functional domain (SHH-N) using microbubble-enhanced sonoporation. A fragment of mouse SHH cDNA encoding SHH-N was subcloned into the mammalian expression vector pcDNA3.1(+) [yielding pcDNA3.1(+)-cSHH]. We confirmed that mouse fibroblasts would produce the SHH-N protein following transfection of pcDNA3.1(+)-cSHH. Application of sonoporation procedures to mouse femoral muscles involves injection of a DNA-microbubble mixture and exposure to ultrasound. A mixture containing pcDNA3.1(+)-cSHH and microbubbles was divided into four doses. The DNA-microbubble mixture was first injected into gelatin capsules in the left femoral muscles and only pcDNA3.1(+) or microbubbles were injected into those on the right. Immediately after injection, ultrasound irradiation was performed utilizing an ultrasound probe placed adjacent to the injected gelatin capsules in femoral muscles. Soft X-ray images were taken at 10, 14, 21, and 28 days. Fourteen days after transduction at doses of 114.75 μg/0.03 ml and 0.2 ml microbubbles, respectively, we observed a non-penetration image, and gradually the non-penetration image was clear until 28 days. Histological observation showed endochondral ossification tissues at 14, 21, and 28 days after transduction at doses of 114.75 μg DNA/0.03 ml microbubbles and 114.75 μg DNA/0.2 ml microbubbles. In the cartilage matrix of proliferative and mature chondrocytes, much SHH was clearly recognized, while in new bone matrix and hypertrophic chondrocytes, BMP-2 was clearly recognized by immunohistochemical staining with SHH and BMP-2 antibody. This is, to our knowledge, the first report describing direct transduction of a SHH-plasmid vector to the muscle of adult nude mice using microbubble-enhanced sonoporation. We confirmed that it is a promising type of gene transduction judging from its simplicity and small amount of injury.

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