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795. Hydrodynamic Gene Delivery to Swine Skeletal Muscle Using Image-Guided, Computer-Controlled Injection System
PHYSICAL METHODS OF DELIVERY of the widely employed adeno-associated virus (AAV) and lentiviral vectors. Two transposable reporter gene segments of 7 and 12 kb in size were constructed, which expressed the green-fluorescent protein fused to a neomycin phosphotransferase gene (GFP-Neo) under the transcriptional control of the cytomegalovirus promoter (CMV). The sizes of the transposable reporter segments were varied using “spacer” fragments obtained from non-coding DNA sequences. HSVT-GFPNeo_7kb and HSVT-GFP-Neo_12kb, when independently codelivered with a SB-expressing HSV amplicon vector (HSV-SB10), generated similar numbers of G418-resistant, eGFP-positive colonies in standard colony formation assays. Overall, our results indicate that the SB transposase is competent in stably integrating transgenon units of at least 12 kb in size within the human genome upon delivery via HSV amplicons. Future experiments will determine the upper bounds of amplicon mediated transgenon integration, detail the characteristics of the amplicon-vector genome that facilitate transposition of such large DNA segments as compared to plasmid-based modalities, and also the distribution of integration sites and their relationship to host cell-expressed genes.
Physical Methods of Delivery 795. Hydrodynamic Gene Delivery to Swine Skeletal Muscle Using Image-Guided, ComputerControlled Injection System
Kenya Kamimura,1 Takeshi Suda,2 Guisheng Zhang,1 Dexi Liu.1 Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA; 2Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan. 1
Computer-controlled injection device for hydrodynamic gene delivery in large animals has been developed1 and proven effective in gene delivery to pig liver2. To extend the application of the hydrodynamic procedure for gene delivery, we evaluated a method of image-guided hydrodynamic gene delivery to skeletal muscle in pigs. The experimental procedure involves an image-guided insertion of a balloon catheter into the femoral vein of the large proximal hind limb from the jugular vein, inflation of the balloon to block the blood flow, and a retrograde hydrodynamic injection of reporter plasmids using a computer-controlled injection device. We targeted the proximal hind limb because of its larger size of the muscle group compared to that of the distal hind limb which was focused by a number of previous studies injecting from the peripheral vein. With the exception of a small incision made at animal neck for catheter insertion, our procedure did not involve any other surgical procedures. We demonstrate that transfection efficiency of the procedure is directly related to the injection pressure, injection volume, and flow rate. The optimal hydrodynamic gene delivery was achieved at injection pressure of 300 psi with an 8 Fr size balloon catheter and flow rate of 15 ml/sec. Under this condition, a continuous injection for 20 sec resulted in an injection volume of 300 ml of plasmid solution (pCMV-Luc, 100 µg/ml in saline) and an increase of intravenous pressure from 20 to 40 mmHg. Among the muscle groups examined 5 days after hydrodynamic gene delivery, the vastus lateralis, biceps femoris and gracilis muscle showed the highest level of luciferase activity at 107 RLU/mg, followed by the sartorius and semimembranous muscle at 106 RLU/mg level. The lowest gene expression was seen in rectus femoris muscle at 105 RLU/mg level. Result from immunohistochemical analysis revealed persistent reporter gene expression for 2 months (length of experiment) in transfected cells. Full blood test at different time points and histological analysis showed no obvious tissue damage with an exception of transient edema that was resolved within 24 hrs after the injection. These results suggest that the image-guided, intravenous hydrodynamic gene delivery to skeletal muscle is effective, safe Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
and site specific. This work was supported in part by NIH grant EB 007357. Reference 1. Takeshi Suda, Kieko Suda and Dexi Liu (2008) Computer-assisted Hydrodynamic Gene Delivery. Mol Ther 16:10981104. 2. Kenya Kamimura, Takeshi Suda, Wei Xu, Guisheng Zhang and Dexi Liu. Image-guided, Lobe-specific Hydrodynamic Gene Delivery to Swine Liver. Mol Ther Advance Online Publication, January 20, 2009; doi:10.1038/mt.2008.294.
796. In Vivo Electroporation of BMP-9 Gene Induces Bone Regeneration and Non-Union Fracture Repair
Gadi Pelled,1,3 Nadav Kimelman-Bleich,1 Yoram Zilberman,1 Jin Z. Li,2 Gregory A. Helm,2 Dan Gazit.1,3 1 Skeletal Biotech Lab, The Hebrew University, Jerusalem, Israel; 2University of Virginia Health System, Charlottesville, VA; 3Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA. Multi-fractures and non-union fractures are major challenges with no optimal solution in orthopedics up to date. Recent findings indicate that in vivo DNA electrotransfer or electroporation (use of short high-voltage pulses to overcome the barrier of the cell membrane) is a safe, simple, and effective technique of gene delivery. We therefore hypothesized that direct in vivo electroporation of an osteogenic gene (BMP-9) into a non-union radius bone defect site would induce complete bone regeneration and fracture repair. A 1.5 mm segmental defect was created in the radius bone of C3H/HeN mice. A 1.5 mm long collagen sponge was implanted in the defect site. Ten days later needle electrodes were placed under fluoroscopic guidance in the defect site, such as that the implanted sponge was located between the electrodes. 50µg of pLuc (n=5) or pBMP9 (n=5) in 11µl of PBSx1 were injected into the collagen sponge. Eight pulses at 100V were generated using an ECM 830 electroporator (BTX). Each pulse was of 20ms with 100ms interval (Figure 1). Five weeks post electroporation the mice were sacrificed, and the dissected limbs were subjected to high-resolution µCT scanning which enabled quantitative analysis of bone formation at the site of the defect.
Our results showed that massive bone formation was evident in the defect site electroporated with pBMP9, based on the µCT analysis (Fig. 2a and c). The new bone appeared to be fused to the defect edges, achieving full bridging of the bone gap. No bone formation was noted in limbs electroporated with pLuc, 5 weeks post electroporation. Some bone growth could be noted in the defect edges, but the defect remained un-bridged (Fig. 2b).
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Physical Methods of Delivery 795. Hydrodynamic Gene Delivery to Swine Skeletal Muscle Using Image-Guided, ComputerControlled Injection System
Kenya Kamimura,1 Takeshi Suda,2 Guisheng Zhang,1 Dexi Liu.1 Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA; 2Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan. 1
Computer-controlled injection device for hydrodynamic gene delivery in large animals has been developed1 and proven effective in gene delivery to pig liver2. To extend the application of the hydrodynamic procedure for gene delivery, we evaluated a method of image-guided hydrodynamic gene delivery to skeletal muscle in pigs. The experimental procedure involves an image-guided insertion of a balloon catheter into the femoral vein of the large proximal hind limb from the jugular vein, inflation of the balloon to block the blood flow, and a retrograde hydrodynamic injection of reporter plasmids using a computer-controlled injection device. We targeted the proximal hind limb because of its larger size of the muscle group compared to that of the distal hind limb which was focused by a number of previous studies injecting from the peripheral vein. With the exception of a small incision made at animal neck for catheter insertion, our procedure did not involve any other surgical procedures. We demonstrate that transfection efficiency of the procedure is directly related to the injection pressure, injection volume, and flow rate. The optimal hydrodynamic gene delivery was achieved at injection pressure of 300 psi with an 8 Fr size balloon catheter and flow rate of 15 ml/sec. Under this condition, a continuous injection for 20 sec resulted in an injection volume of 300 ml of plasmid solution (pCMV-Luc, 100 µg/ml in saline) and an increase of intravenous pressure from 20 to 40 mmHg. Among the muscle groups examined 5 days after hydrodynamic gene delivery, the vastus lateralis, biceps femoris and gracilis muscle showed the highest level of luciferase activity at 107 RLU/mg, followed by the sartorius and semimembranous muscle at 106 RLU/mg level. The lowest gene expression was seen in rectus femoris muscle at 105 RLU/mg level. Result from immunohistochemical analysis revealed persistent reporter gene expression for 2 months (length of experiment) in transfected cells. Full blood test at different time points and histological analysis showed no obvious tissue damage with an exception of transient edema that was resolved within 24 hrs after the injection. These results suggest that the image-guided, intravenous hydrodynamic gene delivery to skeletal muscle is effective, safe Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
and site specific. This work was supported in part by NIH grant EB 007357. Reference 1. Takeshi Suda, Kieko Suda and Dexi Liu (2008) Computer-assisted Hydrodynamic Gene Delivery. Mol Ther 16:10981104. 2. Kenya Kamimura, Takeshi Suda, Wei Xu, Guisheng Zhang and Dexi Liu. Image-guided, Lobe-specific Hydrodynamic Gene Delivery to Swine Liver. Mol Ther Advance Online Publication, January 20, 2009; doi:10.1038/mt.2008.294.
796. In Vivo Electroporation of BMP-9 Gene Induces Bone Regeneration and Non-Union Fracture Repair
Gadi Pelled,1,3 Nadav Kimelman-Bleich,1 Yoram Zilberman,1 Jin Z. Li,2 Gregory A. Helm,2 Dan Gazit.1,3 1 Skeletal Biotech Lab, The Hebrew University, Jerusalem, Israel; 2University of Virginia Health System, Charlottesville, VA; 3Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA. Multi-fractures and non-union fractures are major challenges with no optimal solution in orthopedics up to date. Recent findings indicate that in vivo DNA electrotransfer or electroporation (use of short high-voltage pulses to overcome the barrier of the cell membrane) is a safe, simple, and effective technique of gene delivery. We therefore hypothesized that direct in vivo electroporation of an osteogenic gene (BMP-9) into a non-union radius bone defect site would induce complete bone regeneration and fracture repair. A 1.5 mm segmental defect was created in the radius bone of C3H/HeN mice. A 1.5 mm long collagen sponge was implanted in the defect site. Ten days later needle electrodes were placed under fluoroscopic guidance in the defect site, such as that the implanted sponge was located between the electrodes. 50µg of pLuc (n=5) or pBMP9 (n=5) in 11µl of PBSx1 were injected into the collagen sponge. Eight pulses at 100V were generated using an ECM 830 electroporator (BTX). Each pulse was of 20ms with 100ms interval (Figure 1). Five weeks post electroporation the mice were sacrificed, and the dissected limbs were subjected to high-resolution µCT scanning which enabled quantitative analysis of bone formation at the site of the defect.
Our results showed that massive bone formation was evident in the defect site electroporated with pBMP9, based on the µCT analysis (Fig. 2a and c). The new bone appeared to be fused to the defect edges, achieving full bridging of the bone gap. No bone formation was noted in limbs electroporated with pLuc, 5 weeks post electroporation. Some bone growth could be noted in the defect edges, but the defect remained un-bridged (Fig. 2b).
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