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Sendai Virus Vector Can Efficiently Introduce Transgene into Cardiomyocytes In Vitro and In Vivo
CARDIAC GENE AND CELL THERAPY myocardial gene delivery. In contrast to adenovirus, which has generally been the vector of choice for many heart trials, SV40 does not elicit an immune response allowing repeated administrations, if necessary, to prolong the effect of the delivered gene. Furthermore, SV40 vectors are capable of infecting dividing or non-dividing cells. Our unique vector, prepared in vitro from recombinant SV40 capsid proteins and plasmid DNA, has no viral sequences, providing unique critical safety advantages. Therapeutic DNA of considerable size can be readily packaged into these vectors. We tested the efficiency of SV40 vectors to introduce a reporter gene into myocardial cells, in vitro. Methods: The transduction efficiency of SV40 vectors containing luciferase (SV40/ luc) as a reporter gene was tested on spontaneously beating primary cultures derived from rat neonatal hearts (2.25x106 cells/well). Luciferase avtivity was determined in triplicate 48 hours post infection, after lysing the cells using a SPECTRAFluor Plus microplate luminometer. Cell viability was determined prior to the luciferase assay by Trypan blue exclusion. Results: The dose response to different concentrations of SV40/ luc was tested. Luciferase expression increased with was optimal at 4x107 virus particles/well (Fig.1). No difference in cell viability (89%) was detected between SV40/ luc treated and control cells. Conclusions: These results indicate that our SV40 vector successfully transduces cardiomyocytes without any apparent cardiotoxicity. This suggests that this vector is a promising vehicle for cardiac gene therapy.
using SeVV in the context of myocardial gene therapy was not evaluated. In the present study, we evaluated SeVV-mediated genetransfer into cardiomyocytes in vitro and in vivo. Materials and Methods: Sendai virus vector (SeVV) encoding E.coli β-galactosidase gene (LacZ, SeV/LacZ) was provided by DNAVEC Research Inc. Adenoviral vector encoding LacZ (AxCAZ3) was also used in this study. Rat neonatal cardiomyocytes were isolated from newborn Lewis rat. Briefly, the ventricles obtained from neonatal rat were digested by collagenase (5 mg/ml) for 60 min at 37°C. Isolated naonatal cardiomyocytes were seeded into culture plates and cultured in DMEM supplemented with 10% fetal bovine serum (FBS). Neonatal cardiomyocytes were infected with SeV/ LacZ or AxCAZ3 at various multiplicity of infection (MOI) in DMEM with 2% FBS . We also evaluated the effects of exposure times and the temperature of SeVV infection. LacZ expression in cardiomyocytes was evaluated by a beta-gal reporter gene assay and X-gal staining 24 h after viral infection. For evaluation of SeVV-mediated gene expression in the heart, Lewis rats were anesthetized and the heart was exposed through a left lateral thoracotomy. Various titer of SeV/LacZ or AxCAZ3 was injected intramyocardially into apex of the heart. Rats were sacrified 48 h after gene-transduction and major organs (heart, rt. lung, liver, rt. kidney, spleen) were excised for the evaluation of LacZ expression. Results: Although SeV/LacZ and AxCAZ3 both resulted in equally efficient transgene expression in rat neonatal cardiomyocytes when used at a high MOI, the former was more effective at low MOI. Furthermore, SeVV-mediated gene transduction had several other advantages for myocardial gene therapy, such as a requirement for only a brief exposure time as well as high gene-transduction efficiency at low temperatures in vitro. We also confirmed superior transgene expression in the rat heart and very little extra-cardiac gene expression in the lung and spleen after direct myocardial injection of SeVV. There were no manifestations of respiratory infection at any time in animals receiving SeVV. Conclusion: The unique features of SeVV, namely, highly efficient gene transduction, excellent transgene expression, brief exposure times for infection, and high infectivity at low temperatures, may make this virus the vector of choice for myocardial gene delivery. Thus, the Sendai virus may represent a novel class of viral vector for cardiovascular gene therapy.
933. Sendai Virus Vector Can Efficiently Introduce Transgene into Cardiomyocytes In Vitro and In Vivo
934. Comparing Two Modalities of Myocardial Gene Delivery: Percutaneous Retrograde Coronary Venous Delivery and Intramyocardial Injection
Yukiko Honma,1,2 Yoshinori Ito,1,3 Hironari Dehari,4 Masayoshi Kobune,5 Jinghua Huang,3 Kiminori Nakamura,3 Takeshi Kobayashi,6 Takeshi Uzuka,3 Sachie Hirai,3 Hiroaki Uchida,7 Makoto Inoue,8 Mamoru Hasegawa,8 Masayuki Morikawa,2 Noritsugu Tohse,6 Tomio Abe,2 Hirofumi Hamada.3 1 Gene Therapy, Sapporo Medical University, Sapporoc, Japan; 2 Thoracic and Cardiovascular Surgery, Sapporo Medical University, Sapporo, Japan; 3Molecular Medicine, Sapporo Medical University, Sapporo, Japan; 4Oral Surgery, Sapporo Medical University, Sapporo, Japan; 54th Department of Internal Medicine, Sapporo Medical University, Sapporo, Japan; 6Cellular Physiology and Signal Transduction, Sapporo Medical University, Sapporo, Japan; 7Surgery and Bioengineering, Institute of Medical Science, University of Tokyo, Tokyo, Japan; 8DNAVEC Research Inc., Tsukuba, Japan. Background: Sendai virus (SeV), a rodent respiratory virus considered non-pathogenic for humans, is a negative-strand RNA virus, classified as a type I parainfluenza virus belonging to the family Paramyxoviridae. Recently, efficient gene transduction into various primary cultured cells and tissues by a recombinant Sendai virus vector (SeVV) has been reported. However, the feasibility of Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright The American Society of Gene Therapy
Eyas Al-Shaykh Youssef,1 Ping Zhang,1 Pamela Rogers,1 Patrice Tremble,2 Joe Rokovich,2 Brian Johnstone,1 Keith L. March,1 Dongming Hou.1 1 Indiana University School of Medicine, Indiana Center for Vascular Biology and Medicine, Indianapolis, IN; 2Edwards Lifesciences, CA. Background Myocardial gene therapy is emerging as a novel modality for treating heart disease. However, the optimal technique maximizing safety and efficacy for myocardial gene delivery is not well established. In this study we evaluated two delivery techniques in swine; percutaneous retrograde coronary venous delivery (RCVD) and direct intramyocardial injection (DMI). Methods Yorkshire cross swine (13 animals total) of mixed gender were randomly assigned to 3 treatment groups. RCVD was performed using an end hole balloon catheter placed transvenously into the anterior interventricular coronary vein. 10 ml of β-gal plasmid (1mg/ ml) was injected using either manual high pressure (n = 5) or pressure wire guided-low pressure injections (n = 4). In the DMI group (n = S357
using SeVV in the context of myocardial gene therapy was not evaluated. In the present study, we evaluated SeVV-mediated genetransfer into cardiomyocytes in vitro and in vivo. Materials and Methods: Sendai virus vector (SeVV) encoding E.coli β-galactosidase gene (LacZ, SeV/LacZ) was provided by DNAVEC Research Inc. Adenoviral vector encoding LacZ (AxCAZ3) was also used in this study. Rat neonatal cardiomyocytes were isolated from newborn Lewis rat. Briefly, the ventricles obtained from neonatal rat were digested by collagenase (5 mg/ml) for 60 min at 37°C. Isolated naonatal cardiomyocytes were seeded into culture plates and cultured in DMEM supplemented with 10% fetal bovine serum (FBS). Neonatal cardiomyocytes were infected with SeV/ LacZ or AxCAZ3 at various multiplicity of infection (MOI) in DMEM with 2% FBS . We also evaluated the effects of exposure times and the temperature of SeVV infection. LacZ expression in cardiomyocytes was evaluated by a beta-gal reporter gene assay and X-gal staining 24 h after viral infection. For evaluation of SeVV-mediated gene expression in the heart, Lewis rats were anesthetized and the heart was exposed through a left lateral thoracotomy. Various titer of SeV/LacZ or AxCAZ3 was injected intramyocardially into apex of the heart. Rats were sacrified 48 h after gene-transduction and major organs (heart, rt. lung, liver, rt. kidney, spleen) were excised for the evaluation of LacZ expression. Results: Although SeV/LacZ and AxCAZ3 both resulted in equally efficient transgene expression in rat neonatal cardiomyocytes when used at a high MOI, the former was more effective at low MOI. Furthermore, SeVV-mediated gene transduction had several other advantages for myocardial gene therapy, such as a requirement for only a brief exposure time as well as high gene-transduction efficiency at low temperatures in vitro. We also confirmed superior transgene expression in the rat heart and very little extra-cardiac gene expression in the lung and spleen after direct myocardial injection of SeVV. There were no manifestations of respiratory infection at any time in animals receiving SeVV. Conclusion: The unique features of SeVV, namely, highly efficient gene transduction, excellent transgene expression, brief exposure times for infection, and high infectivity at low temperatures, may make this virus the vector of choice for myocardial gene delivery. Thus, the Sendai virus may represent a novel class of viral vector for cardiovascular gene therapy.
933. Sendai Virus Vector Can Efficiently Introduce Transgene into Cardiomyocytes In Vitro and In Vivo
934. Comparing Two Modalities of Myocardial Gene Delivery: Percutaneous Retrograde Coronary Venous Delivery and Intramyocardial Injection
Yukiko Honma,1,2 Yoshinori Ito,1,3 Hironari Dehari,4 Masayoshi Kobune,5 Jinghua Huang,3 Kiminori Nakamura,3 Takeshi Kobayashi,6 Takeshi Uzuka,3 Sachie Hirai,3 Hiroaki Uchida,7 Makoto Inoue,8 Mamoru Hasegawa,8 Masayuki Morikawa,2 Noritsugu Tohse,6 Tomio Abe,2 Hirofumi Hamada.3 1 Gene Therapy, Sapporo Medical University, Sapporoc, Japan; 2 Thoracic and Cardiovascular Surgery, Sapporo Medical University, Sapporo, Japan; 3Molecular Medicine, Sapporo Medical University, Sapporo, Japan; 4Oral Surgery, Sapporo Medical University, Sapporo, Japan; 54th Department of Internal Medicine, Sapporo Medical University, Sapporo, Japan; 6Cellular Physiology and Signal Transduction, Sapporo Medical University, Sapporo, Japan; 7Surgery and Bioengineering, Institute of Medical Science, University of Tokyo, Tokyo, Japan; 8DNAVEC Research Inc., Tsukuba, Japan. Background: Sendai virus (SeV), a rodent respiratory virus considered non-pathogenic for humans, is a negative-strand RNA virus, classified as a type I parainfluenza virus belonging to the family Paramyxoviridae. Recently, efficient gene transduction into various primary cultured cells and tissues by a recombinant Sendai virus vector (SeVV) has been reported. However, the feasibility of Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright The American Society of Gene Therapy
Eyas Al-Shaykh Youssef,1 Ping Zhang,1 Pamela Rogers,1 Patrice Tremble,2 Joe Rokovich,2 Brian Johnstone,1 Keith L. March,1 Dongming Hou.1 1 Indiana University School of Medicine, Indiana Center for Vascular Biology and Medicine, Indianapolis, IN; 2Edwards Lifesciences, CA. Background Myocardial gene therapy is emerging as a novel modality for treating heart disease. However, the optimal technique maximizing safety and efficacy for myocardial gene delivery is not well established. In this study we evaluated two delivery techniques in swine; percutaneous retrograde coronary venous delivery (RCVD) and direct intramyocardial injection (DMI). Methods Yorkshire cross swine (13 animals total) of mixed gender were randomly assigned to 3 treatment groups. RCVD was performed using an end hole balloon catheter placed transvenously into the anterior interventricular coronary vein. 10 ml of β-gal plasmid (1mg/ ml) was injected using either manual high pressure (n = 5) or pressure wire guided-low pressure injections (n = 4). In the DMI group (n = S357