753. Ectopic Bone Formation In Vivo Induced by Sonoporation: Ultrasound-Based Non Viral Gene Delivery System

PHYSICAL METHODS OF DELIVERY 752. Delivery of Non-Microparticle Naked DNA Vaccine Using Supersonic Flow by a LowPressure Gene Gun Chi-Chen Lin,1 Ying-Chang Wang,2 Men-Chi Yen,1 Ming-Derg Lai.1 1 Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan; 2BioWare Technologies Co., Ltd, Taipei, Taiwan. DNA vaccines are a new and powerful approach to generation of immunological responses against infectious disease and cancer. DNA can be delivered either into muscle by simple injection or into epidermal by gene gun. Intramuscular injection requires large amount of DNA (100microgram/per mouse) to elicit the immune response; in contrast, microparticlulate bombardment system can induce immune response using very low amount of DNA (1 microgram/per mouse). One disadvantage of gene gun bombardment is that nonbiodegradable gold or tungsten may skew the immune response or cause adverse side effect when accumulated. In this report, we have demonstrated the direct delivery of naked DNA without any microparticle using a modified gene gun. The modified gene gun is based on the aerodynamic theory that a supersonic flow is generated when the pressure difference between the inside and the outside pressure of the nozzle is greater than 1.9atm. This high speed airflow can carry the particle from stationary state to accelerate to an extreme high speed (200m/sec). In this way, the naked DNA may be directly transformed into the mammalian cells. Previous study indicated that this modified gene gun achieve similar deliver efficacy as the gene gun commercially available using gold-coated DNA. In this report, we showed that the modified gene gun can achieve 10-40% delivery efficacy as the gold particle coated with DNA in Balb/C mice, C57BL/6 mice, and C3H mice using luciferase gene reporter driven by CMV promoter. Then, we examined the immune responses elicited by inoculation of DNA encoding HBV large surface protein expressed by its own promoter. The non-microparticle DNA and gold-coated DNA elicited similar humoral immunity as shown by antibody titer. Similar overall cytokine profile was induced by either type of DNA vaccine. Furthermore, we examined the therapeutic responses with the DNA vaccine against the extracellular domain of neu on the mouse tumor (MBT-2) naturally overexpressing neu in C3H mice. The results indicated that non-microparticle naked neu DNA vaccine can achieve similar anti-tumor effect as the gold-coated neu DNA vaccine in C3H mice at the dose of 1 microgram/per mouse. Ying-Chang Wang is an employee of BioWare Technologies Co.,Ltd, Taipei, Taiwan.

METHODS: C3H/HeN female mice were injected into the thigh muscle with luciferase (pLuc), human bone morphogenetic protein (phBMP)-9, empty pcDNA3 vector or PBS mixed with echo contrast microbubbles (OptisonTM). Immediately after injection the skin was covered with Aloe-sound gel and transcutaneous, noninvasive sonoporation was applied using 6mm probe of Sonitron2000TM device. In order to monitor transgene expression noninvasively and quantitatively, luciferase activity was detected in vivo in real time using bioluminescence analysis with CCCD. To examine the osteogenesis in vivo, phBMP-9 was sonoporated into the thigh muscle of transgenic mice, stably expressing luciferase gene under osteocalcin (osteogenic marker) promoter (Oc-luc). Qualitative and quantitative analysis of bone formation was performed using three-dimensional (3D) high-resolution micro-CT imaging. RESULTS: In vivo, bioluminescence analysis of luciferase gene expression in sonoporated thigh muscle indicated that the luciferase gene was expressed up to 30 days after sonoporation, with peak expression on day 14 (fig.1a-b). The degree and the duration of luciferase expression were highly correlated to exposure duration of sonoporation (fig.1b). The luciferase expression in sonoporated muscle of Oc-luc transgenic mice indicates the expression of osteocalcin gene, which reflects bone formation process (fig.1c-d). In animals sonoporated with phBMP9 the expression of osteocalcin has been increasing during the first weeks and peaked on day 14, which is the expected profile in bone formation process (fig.1d). Micro-CT imaging with 3D reconstruction revealed ectopic bone formation in the treated thigh muscle (fig.1e). CONCLUSIONS: Sonoporation was found to be an effective non-viral delivery system in vivo. Gene expression lasted up to 4 weeks, and ectopic bone formation was achieved in mice. Thus, applying ultrasound-based gene delivery of osteogenic genes in nonunion bone defects site might have great potential to repair critical size defects and contribute to the development of a clinically applicable solution for currently untreatable bone defects.

753. Ectopic Bone Formation In Vivo Induced by Sonoporation: Ultrasound-Based Non Viral Gene Delivery System Dima Sheyn,1 Gadi Pelled,1 Yoram Zilberman,1 Zulma Gazit,1 Dan Gazit.1 1 Skeletal Biotech Laboratory, Dental Medical School, Hadassah Medical Center, Hebrew University of Jerusalem, Jerusalem, Israel. INTRODUCTION: One of the major obstacles to be addressed by gene therapy approaches in regenerative medicine remains the attainment of an efficient and safe method of gene delivery. The objective of this study was to develop ultrasound-based gene delivery system, called sonoporation, in order to achieve effective gene transfer in vivo and, consequently, bone formation. We hypothesized that direct in vivo sonoporation of naked DNA encoding an osteogenic gene (BMP-9) would induce bone formation.

Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

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