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Modification of plasmid DNA-based gene transfer into central nerve system
International Congress Series 1274 (2004) 159 – 163
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Modification of plasmid DNA-based gene transfer into central nerve system Yoshiaki Taniyamaa,b,*, Munehisa Shimamuraa,c, Naoyuki Satoa,b, Naruya Tomitab, Masayuki Endhoc, Junya Azumaa,b, Kazuma Iekushia,b, Yasufumi Kanedac, Toshio Ogiharab, Ryuichi Morishitaa a
Division of Clinical Gene Therapy, Graduate School of Medicine, Osaka University, Osaka, Japan Department of Geriatric Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan c Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Osaka, Japan
b
Abstract Although viral vector systems are efficient to transfect foreign genes into a variety of tissues, safety issues remain in relation to human gene therapy. In this study, we examined the feasibility of a novel nonviral vector system by using high-frequency, low-intensity ultrasound irradiation for transfection into vascular cells, kidneys and the central nerve systems of fetal mice. As a result, expression of the reporter gene, Venus, was readily detected in the central nervous system. The transfected cells were mainly detected in meningeal cells with intracisternal injection. Overall, the present study demonstrated the feasibility of efficient plasmid DNA transfer into several organs, especially the central nervous system, providing a new option for treating various diseases. D 2004 Elsevier B.V. All rights reserved. Keywords: Ultrasound; Microbubble; Gene therapy; Central nerve system
1. Introduction Gene therapy, based on ultrasound [1] with microbubbles, offers a novel approach for the prevention and treatment of a variety of diseases [2–6]. The major development of * Corresponding author. Division of Clinical Gene Therapy, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, 565-0871 Osaka, Japan. Tel.: +81 6 6879 3406; fax: +81 6 6879 3409. E-mail address: [email protected] (Y. Taniyama). 0531-5131/ D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.07.018
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gene transfer has been its important contribution to intense investigation of the potential of gene therapy in cancer or cardiovascular medicine [2,3]. The amazing advances in molecular biology have provided a dramatic improvement of the technology that is necessary to transfer target genes into somatic cells. Gene transfer methods have surprisingly improved. In fact, some of them (retroviral vectors, adenoviral vectors or liposome-based vectors, etc.) have been used in clinical trials already. However, some severe side effects were reported in clinical gene therapy using such virals, such that people desire safe and efficient clinical gene therapy. Gene therapy for the central nervous system is a promising approach to treat central nervous system diseases. In fact, novel gene therapy has been clinically tried for glioblastom [7,8], Parkinson’s disease [9] and Canavan disease [10]. In these trials, adenovirus and adeno-associated virus (AAV) were commonly used. However, there are serious safety problems, such as immunogenicity [11], delayed demyelination [12] and difficulties in the preparation of a high titer of virus [13], in the case of viral vectors. Since naked plasmid DNA is safe and easy to handle as compared with viral vectors, intramuscular injection of naked plasmid DNA of angiogenic growth factors, such as vascular growth factor (VEGF), has been used clinically for the treatment of ischemic cardiovascular disease [14,15]. Although some researchers have tried to apply direct injection of naked plasmid DNA into the brain [16,17], the transfection efficiency was quite low [16] and a large amount of naked plasmid DNA was required to produce the target protein [17]. Recently ultrasound-mediated gene transfer has been reported to augment the transfection efficiency and facilitate local gene expression [6]. Interestingly, gene transfer into the fetal central nervous system was successfully achieved by intrauterine injection with microbubble-enhanced ultrasound [2,3]. Compared with other viral vectors, there are some theoretical advantages including safety, simplicity of preparation and local gene transfer. Thus, we focused on the development of gene transfer using naked plasmid DNA with an ultrasound or microbubble-enhanced ultrasound method. In the present study, we investigated the possibility of improving plasmid DNA-based gene transfer into the rat brain. 2. Vascular cells We initially examined the transfection efficiency of naked luciferase gene plasmid by three different methods. Transfection of naked luciferase plasmid DNA exhibited low transfection efficiency in human endothelial cells at 1 day after transfection. Transfection of plasmid DNA by ultrasound resulted in a significant increase in luciferase activity ( Pb0.01), and transfection with ultrasound and Optison resulted in a high increase in luciferase activity compared with ultrasound alone ( Pb0.01). Next, we performed electron microscopic scanning of cells transfected with plasmid DNA by using ultrasound with Optison. Immediately after transfection, both endothelial cells and VSMC exhibited small holes in the cell surface and eventually returned to a normal appearance within 24 h. It is suspected that ultrasound irradiation with Optison causes transient holes in the cell surface, thereby resulting in rapid translocation of plasmid
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DNA from outside into the cytoplasm. Ultrasound alone appears to cause smaller holes as compared with coadministration of Optison. No holes were detected in untransfected cells or cells under Optison alone [2,3]. 3. Kidney To transfect NFnB-decoy, we employed a novel approach using ultrasound exposure with an echocardiographic contrast agent, Optison, and clearly demonstrated successful transfection of NFnB-decoy into renal tissue. The therapeutic effect of NFnB-decoy on renal allografts was then evaluated in a rat renal allograft model (Wistar–Lewis). In the control group, graft function significantly deteriorated with marked destruction of renal tissue, accompanied by increased production of major inflammatory mediators, and all of the animals died of renal failure by 9 days. In contrast, graft function (serum creatinine on day 2, NFnB-treated: 0.97F0.16 vs. control: 1.84F0.23 mg/dl, Pb0.01) and histological structure were well preserved with significantly decreased expression of NFnB-regulated cytokines and adhesion molecules, including IL-1, iNOS, MCP-1, TNFa and ICAM-1, in allografts transfected with NFnB-decoy. As a result, animal survival was significantly prolonged in this group as compared with controls (14.2F5.2 vs. 7.1F1.2 days, Pb0.01) [4]. 4. Fetal mice Intrauterine injection of naked DNA expressing luciferase, green fluorescent protein (GFP) or h-galactosidase (h-gal) and fluorescein isothiocyanate-labeled oligodeoxynucleotide (FITC-ODN) in combination with microbubble-enhanced ultrasound (US) produced high-level protein expression in fetal mice. Using ultrasound with Optison, luciferase expression increased approximately 10-fold in comparison to expression after injection of naked DNA alone. Widespread expression of GFP was observed in multiple fetal tissues adjacent to the injection points. Thus, ultrasound with Optison might provide a useful means to clarify the molecular mechanisms of genetic diseases in utero, as well as a tool to develop gene therapies in utero [6]. 5. Central nerve system 5.1. Method 5.1.1. Preparation of naked DNA-microbubble mixture Super-coiled plasmid DNA was dissolved in TE. Optison (Mallinckrodt, San Diego, CA, USA) was used for microbubbles. The plasmid-microbubble solution was prepared just before injection. For intracisternal injection, a total volume of 100 Al containing 200 Ag plasmid and Optison (0, 6.25, 12.5, 25 or 50 Al) was used. For intrastriatum injection, a total volume of 6 Al containing 50 Ag plasmid and Optison (1.5 Al) was used. 5.1.2. In vivo gene transfer in normal rats Male Wistar rats (230–260 g; Charles River Japan, Atsugi, Japan) were used in this study. For infusion into the subarachnoid space, the head of each animal was fixed in a prone position and the atlanto-occipital membrane was exposed through an occipitocerebral
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midline incision. A stainless steel cannula was introduced into the cisterna magna (subarachnoid space). Plasmid DNA solution was infused at 50 Al/min using an injector (IM-3, Narishige Scientific Instrument Laboratory, Tokyo, Japan) after removing 100 Al CSF. Then, animals were exposed to ultrasound from the injection site or temporal bone. For infusion into the striatum, animals were placed in a stereotactic frame (Narishige Scientific Instrument Laboratory), with the skull exposed. A stainless steel cannula was introduced into the striatum. Plasmid DNA solution was injected at 2.0 Al/min using the injector. Rats were kept in the same position for 3 min to avoid loss of plasmid by backflow. Then the animals were insonated from the parietal bone. The ultrasound-emitting transducer used in the present study was an Ultax UX-301 (Celcom, Fukuoka, Japan) with contact gel. The ultrasound parameters were as described previously with minor modification: the transducer diameter was 29 mm, the center frequency was 1-MHz continuous wave (CW), the duty cycle was 26% (on cycles/off cycles=26/74, pulse repetition frequency=2Hz), the intensity was up to 5 W/cm2 and acoustic pressure was 0.55 MPa. Animals were exposed once in each experiment immediately after injection of naked DNA. 5.2. Result First, we measured luciferase activity in the brainstem and cerebellum after injection of the luciferase gene into the cisterna magna. Although luciferase activity could be detected following injection of naked DNA alone, it was very low. After injection of naked DNA, insonation without microbubbles, Optison, showed a tendency to increase luciferase activity. As expected, luciferase activity was markedly enhanced by the addition of 25% Optison and ultrasound, while addition of Optison alone without insonation did not affect luciferase activity. Second, to examine which cells could be transfected, we transfected the Venus gene into the cisterna magna. Different from other vectors, such as adenovirus, gene expression was limited to the brainstem and cerebellum where insonation was performed. There was no gene expression in the cerebral surface, striatum or choroid plexus in the lateral ventricle. Coronal sections of the brainstem showed readily detectable fluorescence of Venus in meningeal cells in the pia mater and arachnoid membrane. 6. Conclusion Overall, the present study demonstrated a safe and efficient method of nonviral gene transfer into the adult central nervous system. A novel therapeutic strategy using plasmid DNA of neurotrophic factors, such as HGF, FGF or BDNF with Optison and ultrasound, is likely to create new therapeutic options in the treatment of Parkinson’s disease and tumors. References [1] K. Tachibana, et al., Induction of cell-membrane porosity by ultrasound, Lancet 353 (9162) (1999 Apr. 24) 1409. [2] Y. Taniyama, et al., Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle, Gene Ther. 9 (6) (2002 Mar.) 372 – 380.
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[3] Y. Taniyama, et al., Local delivery of plasmid DNA into rat carotid artery using ultrasound, Circulation 105 (10) (2002 Mar. 12) 1233 – 1239. [4] H. Azuma, et al., Transfection of NFkB decoy oligodeoxynucleotides using high efficient ultrasoundmediated gene transfer into the donor kidney prolonged survival of rat renal allograft, Gene Ther. 10 (2003) 415 – 425. [5] H.Y. Lan, et al., Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasoundmicrobubble system in rat UUO model, J. Am. Soc. Nephrol. 14 (6) (2003 Jun.) 1535 – 1548. [6] M. Endoh, et al., Fetal gene transfer by intrauterine injection with microbubble-enhanced ultrasound, Molec. Ther. 5 (2002 May) 501 – 508. [7] S.L. Eck, et al., Treatment of recurrent or progressive malignant glioma with a recombinant adenovirus expressing human interferon-beta (H5.010CMVhIFN-beta): a phase I trial, Hum. Gene Ther. 12 (1) (2001 Jan. 1) 97 – 113. [8] T.W. Trask, et al., Phase I study of adenoviral delivery of the HSV-tk gene and ganciclovir administration in patients with current malignant brain tumors, Molec. Ther. 1 (2) (2000 Feb.) 195 – 203. [9] M.J. During, et al., Subthalamic GAD gene transfer in Parkinson disease patients who are candidates for deep brain stimulation, Hum. Gene Ther. 12 (2001) 1589 – 1591. [10] C. Janson, et al., Clinical protocol. Gene therapy of Canavan disease: AAV-2 vector for neurosurgical delivery of aspartoacylase gene (ASPA) to the human brain, Hum. Gene Ther. 13 (11) (2002 Jul. 20) 1391 – 1412. [11] M.A. Schnell, et al., Activation of innate immunity in nonhuman primates following intraportal administration of adenoviral vectors, Molec. Ther. 3 (5 Pt. 1) (2001 May) 708 – 722. [12] R.A. Dewey, et al., Chronic brain inflammation and persistent herpes simplex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-mediated gene therapy: implications for clinical trials, Nat. Med. 5 (11) (1999 Nov.) 1256 – 1263. [13] G. Hsich, M. Sena-Esteves, X.O. Breakefield, Critical issues in gene therapy for neurologic disease, Hum. Gene Ther. 13 (2002) 579 – 604. [14] I. Baumgartner, et al., Lower-extremity edema associated with gene transfer of naked DNA encoding vascular endothelial growth factor, Ann. Intern. Med. 132 (11) (2000 Jun. 6) 880 – 884. [15] T.K. Rosengart, et al., Angiogenesis gene therapy: phase I assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA to individuals with clinically significant severe coronary artery disease, Circulation 100 (5) (1999 Aug. 3) 468 – 474. [16] B. Schwartz, et al., Gene transfer by naked DNA into adult mouse brain, Gene Ther. 3 (5) (1996 May) 405 – 411. [17] K. Sekiguchi, F. Yasuzumi, R. Morishita, Exogenous expression of hepatocyte growth factor (HGF) in rat striatum by naked plasmid DNA, Neurosci. Res. 45 (2003) 173 – 180.
www.ics-elsevier.com
Modification of plasmid DNA-based gene transfer into central nerve system Yoshiaki Taniyamaa,b,*, Munehisa Shimamuraa,c, Naoyuki Satoa,b, Naruya Tomitab, Masayuki Endhoc, Junya Azumaa,b, Kazuma Iekushia,b, Yasufumi Kanedac, Toshio Ogiharab, Ryuichi Morishitaa a
Division of Clinical Gene Therapy, Graduate School of Medicine, Osaka University, Osaka, Japan Department of Geriatric Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan c Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Osaka, Japan
b
Abstract Although viral vector systems are efficient to transfect foreign genes into a variety of tissues, safety issues remain in relation to human gene therapy. In this study, we examined the feasibility of a novel nonviral vector system by using high-frequency, low-intensity ultrasound irradiation for transfection into vascular cells, kidneys and the central nerve systems of fetal mice. As a result, expression of the reporter gene, Venus, was readily detected in the central nervous system. The transfected cells were mainly detected in meningeal cells with intracisternal injection. Overall, the present study demonstrated the feasibility of efficient plasmid DNA transfer into several organs, especially the central nervous system, providing a new option for treating various diseases. D 2004 Elsevier B.V. All rights reserved. Keywords: Ultrasound; Microbubble; Gene therapy; Central nerve system
1. Introduction Gene therapy, based on ultrasound [1] with microbubbles, offers a novel approach for the prevention and treatment of a variety of diseases [2–6]. The major development of * Corresponding author. Division of Clinical Gene Therapy, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, 565-0871 Osaka, Japan. Tel.: +81 6 6879 3406; fax: +81 6 6879 3409. E-mail address: [email protected] (Y. Taniyama). 0531-5131/ D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.07.018
160
Y. Taniyama et al. / International Congress Series 1274 (2004) 159–163
gene transfer has been its important contribution to intense investigation of the potential of gene therapy in cancer or cardiovascular medicine [2,3]. The amazing advances in molecular biology have provided a dramatic improvement of the technology that is necessary to transfer target genes into somatic cells. Gene transfer methods have surprisingly improved. In fact, some of them (retroviral vectors, adenoviral vectors or liposome-based vectors, etc.) have been used in clinical trials already. However, some severe side effects were reported in clinical gene therapy using such virals, such that people desire safe and efficient clinical gene therapy. Gene therapy for the central nervous system is a promising approach to treat central nervous system diseases. In fact, novel gene therapy has been clinically tried for glioblastom [7,8], Parkinson’s disease [9] and Canavan disease [10]. In these trials, adenovirus and adeno-associated virus (AAV) were commonly used. However, there are serious safety problems, such as immunogenicity [11], delayed demyelination [12] and difficulties in the preparation of a high titer of virus [13], in the case of viral vectors. Since naked plasmid DNA is safe and easy to handle as compared with viral vectors, intramuscular injection of naked plasmid DNA of angiogenic growth factors, such as vascular growth factor (VEGF), has been used clinically for the treatment of ischemic cardiovascular disease [14,15]. Although some researchers have tried to apply direct injection of naked plasmid DNA into the brain [16,17], the transfection efficiency was quite low [16] and a large amount of naked plasmid DNA was required to produce the target protein [17]. Recently ultrasound-mediated gene transfer has been reported to augment the transfection efficiency and facilitate local gene expression [6]. Interestingly, gene transfer into the fetal central nervous system was successfully achieved by intrauterine injection with microbubble-enhanced ultrasound [2,3]. Compared with other viral vectors, there are some theoretical advantages including safety, simplicity of preparation and local gene transfer. Thus, we focused on the development of gene transfer using naked plasmid DNA with an ultrasound or microbubble-enhanced ultrasound method. In the present study, we investigated the possibility of improving plasmid DNA-based gene transfer into the rat brain. 2. Vascular cells We initially examined the transfection efficiency of naked luciferase gene plasmid by three different methods. Transfection of naked luciferase plasmid DNA exhibited low transfection efficiency in human endothelial cells at 1 day after transfection. Transfection of plasmid DNA by ultrasound resulted in a significant increase in luciferase activity ( Pb0.01), and transfection with ultrasound and Optison resulted in a high increase in luciferase activity compared with ultrasound alone ( Pb0.01). Next, we performed electron microscopic scanning of cells transfected with plasmid DNA by using ultrasound with Optison. Immediately after transfection, both endothelial cells and VSMC exhibited small holes in the cell surface and eventually returned to a normal appearance within 24 h. It is suspected that ultrasound irradiation with Optison causes transient holes in the cell surface, thereby resulting in rapid translocation of plasmid
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DNA from outside into the cytoplasm. Ultrasound alone appears to cause smaller holes as compared with coadministration of Optison. No holes were detected in untransfected cells or cells under Optison alone [2,3]. 3. Kidney To transfect NFnB-decoy, we employed a novel approach using ultrasound exposure with an echocardiographic contrast agent, Optison, and clearly demonstrated successful transfection of NFnB-decoy into renal tissue. The therapeutic effect of NFnB-decoy on renal allografts was then evaluated in a rat renal allograft model (Wistar–Lewis). In the control group, graft function significantly deteriorated with marked destruction of renal tissue, accompanied by increased production of major inflammatory mediators, and all of the animals died of renal failure by 9 days. In contrast, graft function (serum creatinine on day 2, NFnB-treated: 0.97F0.16 vs. control: 1.84F0.23 mg/dl, Pb0.01) and histological structure were well preserved with significantly decreased expression of NFnB-regulated cytokines and adhesion molecules, including IL-1, iNOS, MCP-1, TNFa and ICAM-1, in allografts transfected with NFnB-decoy. As a result, animal survival was significantly prolonged in this group as compared with controls (14.2F5.2 vs. 7.1F1.2 days, Pb0.01) [4]. 4. Fetal mice Intrauterine injection of naked DNA expressing luciferase, green fluorescent protein (GFP) or h-galactosidase (h-gal) and fluorescein isothiocyanate-labeled oligodeoxynucleotide (FITC-ODN) in combination with microbubble-enhanced ultrasound (US) produced high-level protein expression in fetal mice. Using ultrasound with Optison, luciferase expression increased approximately 10-fold in comparison to expression after injection of naked DNA alone. Widespread expression of GFP was observed in multiple fetal tissues adjacent to the injection points. Thus, ultrasound with Optison might provide a useful means to clarify the molecular mechanisms of genetic diseases in utero, as well as a tool to develop gene therapies in utero [6]. 5. Central nerve system 5.1. Method 5.1.1. Preparation of naked DNA-microbubble mixture Super-coiled plasmid DNA was dissolved in TE. Optison (Mallinckrodt, San Diego, CA, USA) was used for microbubbles. The plasmid-microbubble solution was prepared just before injection. For intracisternal injection, a total volume of 100 Al containing 200 Ag plasmid and Optison (0, 6.25, 12.5, 25 or 50 Al) was used. For intrastriatum injection, a total volume of 6 Al containing 50 Ag plasmid and Optison (1.5 Al) was used. 5.1.2. In vivo gene transfer in normal rats Male Wistar rats (230–260 g; Charles River Japan, Atsugi, Japan) were used in this study. For infusion into the subarachnoid space, the head of each animal was fixed in a prone position and the atlanto-occipital membrane was exposed through an occipitocerebral
162
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midline incision. A stainless steel cannula was introduced into the cisterna magna (subarachnoid space). Plasmid DNA solution was infused at 50 Al/min using an injector (IM-3, Narishige Scientific Instrument Laboratory, Tokyo, Japan) after removing 100 Al CSF. Then, animals were exposed to ultrasound from the injection site or temporal bone. For infusion into the striatum, animals were placed in a stereotactic frame (Narishige Scientific Instrument Laboratory), with the skull exposed. A stainless steel cannula was introduced into the striatum. Plasmid DNA solution was injected at 2.0 Al/min using the injector. Rats were kept in the same position for 3 min to avoid loss of plasmid by backflow. Then the animals were insonated from the parietal bone. The ultrasound-emitting transducer used in the present study was an Ultax UX-301 (Celcom, Fukuoka, Japan) with contact gel. The ultrasound parameters were as described previously with minor modification: the transducer diameter was 29 mm, the center frequency was 1-MHz continuous wave (CW), the duty cycle was 26% (on cycles/off cycles=26/74, pulse repetition frequency=2Hz), the intensity was up to 5 W/cm2 and acoustic pressure was 0.55 MPa. Animals were exposed once in each experiment immediately after injection of naked DNA. 5.2. Result First, we measured luciferase activity in the brainstem and cerebellum after injection of the luciferase gene into the cisterna magna. Although luciferase activity could be detected following injection of naked DNA alone, it was very low. After injection of naked DNA, insonation without microbubbles, Optison, showed a tendency to increase luciferase activity. As expected, luciferase activity was markedly enhanced by the addition of 25% Optison and ultrasound, while addition of Optison alone without insonation did not affect luciferase activity. Second, to examine which cells could be transfected, we transfected the Venus gene into the cisterna magna. Different from other vectors, such as adenovirus, gene expression was limited to the brainstem and cerebellum where insonation was performed. There was no gene expression in the cerebral surface, striatum or choroid plexus in the lateral ventricle. Coronal sections of the brainstem showed readily detectable fluorescence of Venus in meningeal cells in the pia mater and arachnoid membrane. 6. Conclusion Overall, the present study demonstrated a safe and efficient method of nonviral gene transfer into the adult central nervous system. A novel therapeutic strategy using plasmid DNA of neurotrophic factors, such as HGF, FGF or BDNF with Optison and ultrasound, is likely to create new therapeutic options in the treatment of Parkinson’s disease and tumors. References [1] K. Tachibana, et al., Induction of cell-membrane porosity by ultrasound, Lancet 353 (9162) (1999 Apr. 24) 1409. [2] Y. Taniyama, et al., Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle, Gene Ther. 9 (6) (2002 Mar.) 372 – 380.
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[3] Y. Taniyama, et al., Local delivery of plasmid DNA into rat carotid artery using ultrasound, Circulation 105 (10) (2002 Mar. 12) 1233 – 1239. [4] H. Azuma, et al., Transfection of NFkB decoy oligodeoxynucleotides using high efficient ultrasoundmediated gene transfer into the donor kidney prolonged survival of rat renal allograft, Gene Ther. 10 (2003) 415 – 425. [5] H.Y. Lan, et al., Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasoundmicrobubble system in rat UUO model, J. Am. Soc. Nephrol. 14 (6) (2003 Jun.) 1535 – 1548. [6] M. Endoh, et al., Fetal gene transfer by intrauterine injection with microbubble-enhanced ultrasound, Molec. Ther. 5 (2002 May) 501 – 508. [7] S.L. Eck, et al., Treatment of recurrent or progressive malignant glioma with a recombinant adenovirus expressing human interferon-beta (H5.010CMVhIFN-beta): a phase I trial, Hum. Gene Ther. 12 (1) (2001 Jan. 1) 97 – 113. [8] T.W. Trask, et al., Phase I study of adenoviral delivery of the HSV-tk gene and ganciclovir administration in patients with current malignant brain tumors, Molec. Ther. 1 (2) (2000 Feb.) 195 – 203. [9] M.J. During, et al., Subthalamic GAD gene transfer in Parkinson disease patients who are candidates for deep brain stimulation, Hum. Gene Ther. 12 (2001) 1589 – 1591. [10] C. Janson, et al., Clinical protocol. Gene therapy of Canavan disease: AAV-2 vector for neurosurgical delivery of aspartoacylase gene (ASPA) to the human brain, Hum. Gene Ther. 13 (11) (2002 Jul. 20) 1391 – 1412. [11] M.A. Schnell, et al., Activation of innate immunity in nonhuman primates following intraportal administration of adenoviral vectors, Molec. Ther. 3 (5 Pt. 1) (2001 May) 708 – 722. [12] R.A. Dewey, et al., Chronic brain inflammation and persistent herpes simplex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-mediated gene therapy: implications for clinical trials, Nat. Med. 5 (11) (1999 Nov.) 1256 – 1263. [13] G. Hsich, M. Sena-Esteves, X.O. Breakefield, Critical issues in gene therapy for neurologic disease, Hum. Gene Ther. 13 (2002) 579 – 604. [14] I. Baumgartner, et al., Lower-extremity edema associated with gene transfer of naked DNA encoding vascular endothelial growth factor, Ann. Intern. Med. 132 (11) (2000 Jun. 6) 880 – 884. [15] T.K. Rosengart, et al., Angiogenesis gene therapy: phase I assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA to individuals with clinically significant severe coronary artery disease, Circulation 100 (5) (1999 Aug. 3) 468 – 474. [16] B. Schwartz, et al., Gene transfer by naked DNA into adult mouse brain, Gene Ther. 3 (5) (1996 May) 405 – 411. [17] K. Sekiguchi, F. Yasuzumi, R. Morishita, Exogenous expression of hepatocyte growth factor (HGF) in rat striatum by naked plasmid DNA, Neurosci. Res. 45 (2003) 173 – 180.