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101. Duration of Expression after Gene Electrotransfer to Skin Depends on the Gene of Interest
PHYSICAL METHODS OF DELIVERY 30.50±1.18g injected with pCMV-Adipo or pCMV-Adipo/pCMVAdipoR2 in combination. Animals received pCMV-AdipoR2 had an average body weight of 34.03±1.82g. Body composition analysis using EchoMRI-100TM System revealed that adiponectin and adipoR2 expression in AKR/J mice inhibited the gain of fat mass without change of their lean mass. Serum chemistry tests showed a decrease in treated groups of triglyceride and free fatty acid level. Real time PCR analysis showed a decreased expression of lipogenic genes including FAS and Scd-1 in treated animals. Compared to control animals exhibiting hyperglycemic, hyperinsulinemic and insulin resistance, animals received pCMV-Adipo or pCMV-AdipoR2 plasmids showed normal glucose level and normal insulin sensitivity due to a reduced expression of gluconeogenic enzymes, glucose6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK). These results suggest that over-expression of adiponectin and adiponectin receptor 2 by hydrodynamic gene delivery prevents diet-induced obesity and development of insulin resistance. These results also validate that gene therapy can be an effective approach for managing obesity epidemics.
101. Duration of Expression after Gene Electrotransfer to Skin Depends on the Gene of Interest
Anita Gothelf,1 Jens Eriksen,1 Pernille Hojman,2 Julie Gehl.1 Dept. of Oncology, Copenhagen University Hospital Herlev, Herlev, Denmark; 2Centre of Inammation and Metabolism, Copenhagen University Hospital, Copenhagen, Denmark. 1
Background DNA vaccination is attracting increasing attention and the skin is an interesting target due to easy accessibility and the presence of antigen presenting cells. Gene electrotransfer, or electroporation assisted gene transfer, is a non-viral means of transfecting genes into cells and tissues. It could be ideal for e.g. vaccination purposes, but duration of expression needs to be evaluated to facilitate planning of vaccination trials. Studies with gene electrotransfer with luciferase to skin have shown a peak in expression after 2-3 days, but expression of luciferase could be short-term and may not be the optimal candidate for investigating the kinetics of the transgene expression. We therefore conducted a study using both DNA coding for a uorescent marker molecule (Katushka), and DNA coding for a therapeutic protein (erythropoietin, EPO). This allowed for an evaluation of the duration of expression by a combined visualization of local gene expression and measurement of serum levels of a therapeutic protein. Materials and methods Gene electrotransfer with EPO: Female NMRI mice received either (A) 2 intradermal (i.d.) injections of 50 µg EPO plasmid and subsequent electroporation, (B) 2 i.d. injections of 50 µg EPO plasmid alone, or (C) 2 i.d. injections of 100 µl PBS and subsequent electroporation. At different time points mice were exsanguinated and blood samples were analyzed for serum EPO by ELISA. Gene electrotransfer with katushka: Mice received 1 i.d. injection of 100 µg Katushka plasmid and were subsequently electroporated. Using an Optix MX-2 Time Domain Optical Imaging system (Advanced Research Technologies, Montreal, Canada) mice were scanned after 2, 4, 7, 11, 14, and 21 days. The scans were evaluated by peak of intensity in normalized counts (NC) and the uorescence lifetime. Results Gene electrotransfer with EPO: 24 hours after the transfection the serum EPO level in the group treated with EPO injections and electroporation (group A) serum EPO increased 4.2-fold (p < 0.001) compared to controls, which consistently had serum EPO levels at the baseline. The increase in serum EPO remained statistically signicant until a peak at 1200 pg/ml was reached after 2 weeks. After 4 weeks serum EPO had normalized. Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
Gene electrotransfer with katushka: An increase in intensity was observed after 2 days and peaked after 9 days (2545 NC). After 3 weeks the intensity level was returned to pre-treatment values. The uorescence lifetime was 2.1 ns, which is characteristic for Katushka. Conclusion We have dened the window of transgene expression in mouse skin using two independent methods; a uorescent marker and a protein relevant for systemic therapy. The expression peaks between 9 and 14 days and is normalized after 3 weeks or more. This peak in expression may be different than results achieved after gene electrotransfer to skin with luciferase and states that expression in skin is longer than previously observed. This prole of duration could be of great interest for DNA vaccination to skin in which a sustained production of antigens could be an advantage in eliciting an immune response.
102. Intravenous Delivery of pDNA and siRNA into Muscle by Ultrasound-Mediated Bubble Liposomes
Yoichi Negishi,1 Yoko E. Takahashi,1 Yuko Ishii,1 Ryo Suzuki,2 Kazuo Maruyama,2 Yukihiko Aramaki.1 1 Department of Drug and Gene Delivery System, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan; 2Department of Pharmaceutics, Teikyo University, Sagamihara, Kanagawa, Japan.
Background: Skeletal muscle is an attractive target tissue for numerous gene therapy strategies. Gene delivery for muscle has been extensively studied. Of these, intravascular delivery of naked pDNA is desirable. Muscle has a high density of capillaries that are in close contact with the myobers. Previously, we have developed the polyethyleneglycol (PEG)-modied liposomes entrapping echocontrast gas known as Ultrasound (US) imaging gas. We have called the liposomes “Bubble liposomes” (BLs). It has been reported that BLs improve the tissue permeability by cavitation with US exposure. Here, we modied the naked pDNA or siRNA transfer method into hind limb muscle through blood vessels using BLs and US. Methods: A tourniquet was placed on the upper hind limb to restrict blood ow into and out of the hind limb. pCMV-Luc encoding Luciferase gene and/or its siRNA was injected with BLs into great saphenous vein. Immediately, the muscle was exposed with Ultrasound (Frequency: 1 MHz, Duty: 50 %, time: 120 sec) and subsequently, tourniquet was removed. The limb muscles were harvested and separated at different time point after the gene delivery. The luciferase expressions were measured. Results: Intravenous delivery of pDNA into the muscle can be greatly enhanced when the pDNA was delivered in the combination of BLs and US. In addition, the expression of pDNA was high in the US-focused site. Moreover, the efcient gene delivery can be achieved by intravenous delivery of pDNA into muscle with Bubble liposomes and ultrasound. The expression was also down-regulated by delivering of its siRNA with BLs and US. Conclusion: This US-mediated BLs technique through vein may provide an effective method for gene therapy. Acknowledgements: This study was supported by Industrial Technology Research Grand Program (04A05010) from New Energy and Industrial Technology Development Organization (NEDO) of Japan and Grant-in-Aid for Scientic Research (B) (20300179) from the Japan Society for the Promotion of Science.
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101. Duration of Expression after Gene Electrotransfer to Skin Depends on the Gene of Interest
Anita Gothelf,1 Jens Eriksen,1 Pernille Hojman,2 Julie Gehl.1 Dept. of Oncology, Copenhagen University Hospital Herlev, Herlev, Denmark; 2Centre of Inammation and Metabolism, Copenhagen University Hospital, Copenhagen, Denmark. 1
Background DNA vaccination is attracting increasing attention and the skin is an interesting target due to easy accessibility and the presence of antigen presenting cells. Gene electrotransfer, or electroporation assisted gene transfer, is a non-viral means of transfecting genes into cells and tissues. It could be ideal for e.g. vaccination purposes, but duration of expression needs to be evaluated to facilitate planning of vaccination trials. Studies with gene electrotransfer with luciferase to skin have shown a peak in expression after 2-3 days, but expression of luciferase could be short-term and may not be the optimal candidate for investigating the kinetics of the transgene expression. We therefore conducted a study using both DNA coding for a uorescent marker molecule (Katushka), and DNA coding for a therapeutic protein (erythropoietin, EPO). This allowed for an evaluation of the duration of expression by a combined visualization of local gene expression and measurement of serum levels of a therapeutic protein. Materials and methods Gene electrotransfer with EPO: Female NMRI mice received either (A) 2 intradermal (i.d.) injections of 50 µg EPO plasmid and subsequent electroporation, (B) 2 i.d. injections of 50 µg EPO plasmid alone, or (C) 2 i.d. injections of 100 µl PBS and subsequent electroporation. At different time points mice were exsanguinated and blood samples were analyzed for serum EPO by ELISA. Gene electrotransfer with katushka: Mice received 1 i.d. injection of 100 µg Katushka plasmid and were subsequently electroporated. Using an Optix MX-2 Time Domain Optical Imaging system (Advanced Research Technologies, Montreal, Canada) mice were scanned after 2, 4, 7, 11, 14, and 21 days. The scans were evaluated by peak of intensity in normalized counts (NC) and the uorescence lifetime. Results Gene electrotransfer with EPO: 24 hours after the transfection the serum EPO level in the group treated with EPO injections and electroporation (group A) serum EPO increased 4.2-fold (p < 0.001) compared to controls, which consistently had serum EPO levels at the baseline. The increase in serum EPO remained statistically signicant until a peak at 1200 pg/ml was reached after 2 weeks. After 4 weeks serum EPO had normalized. Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
Gene electrotransfer with katushka: An increase in intensity was observed after 2 days and peaked after 9 days (2545 NC). After 3 weeks the intensity level was returned to pre-treatment values. The uorescence lifetime was 2.1 ns, which is characteristic for Katushka. Conclusion We have dened the window of transgene expression in mouse skin using two independent methods; a uorescent marker and a protein relevant for systemic therapy. The expression peaks between 9 and 14 days and is normalized after 3 weeks or more. This peak in expression may be different than results achieved after gene electrotransfer to skin with luciferase and states that expression in skin is longer than previously observed. This prole of duration could be of great interest for DNA vaccination to skin in which a sustained production of antigens could be an advantage in eliciting an immune response.
102. Intravenous Delivery of pDNA and siRNA into Muscle by Ultrasound-Mediated Bubble Liposomes
Yoichi Negishi,1 Yoko E. Takahashi,1 Yuko Ishii,1 Ryo Suzuki,2 Kazuo Maruyama,2 Yukihiko Aramaki.1 1 Department of Drug and Gene Delivery System, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan; 2Department of Pharmaceutics, Teikyo University, Sagamihara, Kanagawa, Japan.
Background: Skeletal muscle is an attractive target tissue for numerous gene therapy strategies. Gene delivery for muscle has been extensively studied. Of these, intravascular delivery of naked pDNA is desirable. Muscle has a high density of capillaries that are in close contact with the myobers. Previously, we have developed the polyethyleneglycol (PEG)-modied liposomes entrapping echocontrast gas known as Ultrasound (US) imaging gas. We have called the liposomes “Bubble liposomes” (BLs). It has been reported that BLs improve the tissue permeability by cavitation with US exposure. Here, we modied the naked pDNA or siRNA transfer method into hind limb muscle through blood vessels using BLs and US. Methods: A tourniquet was placed on the upper hind limb to restrict blood ow into and out of the hind limb. pCMV-Luc encoding Luciferase gene and/or its siRNA was injected with BLs into great saphenous vein. Immediately, the muscle was exposed with Ultrasound (Frequency: 1 MHz, Duty: 50 %, time: 120 sec) and subsequently, tourniquet was removed. The limb muscles were harvested and separated at different time point after the gene delivery. The luciferase expressions were measured. Results: Intravenous delivery of pDNA into the muscle can be greatly enhanced when the pDNA was delivered in the combination of BLs and US. In addition, the expression of pDNA was high in the US-focused site. Moreover, the efcient gene delivery can be achieved by intravenous delivery of pDNA into muscle with Bubble liposomes and ultrasound. The expression was also down-regulated by delivering of its siRNA with BLs and US. Conclusion: This US-mediated BLs technique through vein may provide an effective method for gene therapy. Acknowledgements: This study was supported by Industrial Technology Research Grand Program (04A05010) from New Energy and Industrial Technology Development Organization (NEDO) of Japan and Grant-in-Aid for Scientic Research (B) (20300179) from the Japan Society for the Promotion of Science.
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