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Enhanced Cardiac Muscle Contractility Achieved by Engineered Zinc Finger Transcriptional Repressors of Phospholamban
CARDIAC GENE AND CELL THERAPY those at the end stage of this disease heart transplantation remains the only and last resort. Acute myocardial infarction (AMI) is a leading cause of heart failure. Increasing evidence suggests that the body has a natural response for cardiac repair by up-regulating stromal cell-derived factor 1 (SDF-1), a stem cell active chemokine, which leads to recruitment of circulating BMSCs to ischemic hearts. However, up-regulation of endogenous SDF-1 is transient and inadequate to induce functionally meaningful heart regeneration. Therefore, the current study aimed to enhance stem cell-initiated cardiac repair by targeting circulating BMSCs to ischemic myocardium through targeted and regulated SDF-1 gene expression. To accomplish this goal, we have developed a regulatable system that consists of a MLC-2v promoter for cardiac selective expression as well as an oxygen-sensitive gene switch based on the oxygendependent degradation domain of hypoxia inducible factor 1α. It delivers genes of interest in a cardiac-selective and hypoxia-regulated manner in vitro and in vivo. AMI was surgically induced in adult BALB/c mice. DNA plasmid encoding hypoxia-regulated human SDF-1 was administered via intra-myocardial injection. PKH26 labeled BMSCs (6 × 106) were systemically injected immediately after induction of AMI. hSDF-1 expression was evaluated by Western blot analysis and the efficiency of hSDF-1 to attract implanted BMSCs was assessed by immunofluorescent staining and confocal microscopy. 1 week post AMI, hSDF-1 expression was markedly increased in the ischemic mouse heart, while less or no expression was detected in spleen, liver, lung, kidney and skeletal muscle nor in the heart of non-MI group. Confocal microscopy showed that hSDF-1 was highly expressed in the peri-infarct zone, with PKH26-labeled BMSCs enriched nearby. Furthermore, infarct size and cardiac fibrosis were significantly reduced in mice treated with hSDF-1 plus BMSCs compared to BMSCs alone. Taken together, our results indicate that cardiac-selective and hypoxiainducible SDF-1 gene transfer activated mobilization of BMSCs to ischemic hearts and facilitated post-ischemic repair. This combined approach of regulated gene transfer and adult stem cells may provide a novel, safe and effective strategy for regenerative medicine, leading to efficient and timely repair of injured tissues.
930. Muscle Stem Cells Deliver Dystrophin and Adopt a Cardiac Phenotype through Both Differentiation and Fusion in the Dystrophic (mdx) Heart Thomas R. Payne,1 Hideki Oshima,2 Tetsuro Sakai,3 Yiqun Ling,2 Burhan Gharabeih,5 James H. Cummins,5 Johnny Huard.1,4,5 1 Bioengineering; 2Cardiothoracic Surgery; 3Anesthesiology; 4 Molecular Genetic and Biochemistry; 5Orthopaedic Surgery, University of Pittsburgh, Children’s Hospital of Pittsburgh, Pittsburgh, PA. A reduction or absence of dystrophin protein in the heart will cause cardiac muscle degeneration and the progressive development of cardiomyopathy. In this study, we investigated the ability of a population of murine skeletal muscle–derived stem cells (MDSCs) to deliver dystrophin and acquire a cardiac phenotype after transplantation into the dystrophic (mdx) murine heart. The injected MDSCs engrafted and partially restored dystrophin expression within the dystrophic (mdx) myocardium for up to 12 weeks after implantation. In these hearts, approximately 3-5% of the donorlabeled cells expressed a cardiac phenotype, and more than half of these cells displayed a hybrid cardiac and skeletal muscle phenotype at 2, 4, 8 and 12 weeks after implantation. The majority of the donor cells expressing a cardiac phenotype were located along the periphery of the graft or within the host myocardium. Transplantation of female MDSCs into male recipient hearts revealed that the injected cells acquired a cardiac phenotype through both differentiation and fusion with host cardiomyocytes. The formation S356
of fibrotic tissue within the injection site may have inhibited further acquisition of a cardiac phenotype by the implanted MDSCs. The results presented here support the potential use of MDSCs to advance muscle cell–based therapies for cardiac repair.
931. Enhanced Cardiac Muscle Contractility Achieved by Engineered Zinc Finger Transcriptional Repressors of Phospholamban H. Steven Zhang,1 Lei Zhang,1 Yuxin Liang,1 Reed Hickey,2 Dmitry Guschin,1 Simon Chandler,1 Mike Kunis,1 Linda Hinh,1 Dengfeng Xia,1 Xiaohong Zhong,1 S. Kaye Spratt,1 J. Tyler Martin,1 Casey C. Case,1 Frank J. Giordano,2 Philip D. Gregory,1 Edward J. Rebar.1 1 Sangamo BioSciences, Richmond, CA; 2Yale University, School of Medicine, New Haven, CT. Phospholamban (PLN) is a critical regulator of cardiac calcium homeostasis and muscle contractility. Recent studies have demonstrated that ablation or inhibition of PLN function can improve cardiac contractile properties in animal models of congestive heart failure (CHF), suggesting that PLN is an attractive target for treatment of CHF. Because of its use of a large, relatively flat interaction surface when docking with its regulation target (Sarcoplasmic Reticulum Ca2+ ATPase 2 isoform A (SERCA2a)), the development of small-molecule inhibitors of PLN function is expected to be extremely challenging. Therefore, approaches that aim to block the expression of PLN may provide a superior means of achieving the desired therapeutic effect. As part of a therapeutic program in CHF we are developing designed zinc finger protein (ZFP) transcriptional repressors of PLN. We have engineered ZFP repressors that target either the human or rat PLN promoter. The rat-specific ZFP repressor gave >90% reduction of PLN mRNA level in a rat heart-derived cell line; and the ZFP repressor targeted to the human PLN promoter produced a similar level of repression in human smooth muscle cells. Microarray analyses indicated that both the rat- and humantargeted ZFPs operated with unrivaled specificity, with PLN being the only gene that was significantly repressed within the monitored genome (14,000-16,000 transcripts). Furthermore, when the rat PLN repressor was introduced into primary cardiomyocytes of day 1 neonates, it efficiently repressed PLN transcription, despite the high level of PLN expression in these cells. Most importantly, when the same ZFP was introduced into adult rat hearts, subsequently isolated ZFP-positive myocytes showed accelerated calcium transients as well as improved contractility, highlighting the functional significance of ZFP mediated PLN repression. Our results demonstrate that, by repressing PLN transcription, engineered ZFPs can enhance the contractile properties of cardiac myocytes, and therefore have the potential of improving cardiac function in CHF patients. The ability to design therapeutically relevant ZFPs can be applied to alter the transcription programs of many disease-related genes. Furthermore, the highly effective and specific ZFP-based approaches provide an alternative to technologies such as small interfering RNA (siRNA), and offer versatility that is unmatched by cDNA-based gene therapies.
932. SV40 Pseudovirions Are Efficient and Safe Myocardial Gene Therapy Vectors Ronen Beeri,1 Thea Pugatsch,1 Suzane Abedat,1 Mahmud Abdel Latif,2 Ariella Oppenheim.2 1 Heart Institute, Hadassah-Hebrew University Medical Center, Jerusalem, Israel; 2Hematology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel. Background: Simian virus-40 (SV40) is an efficient vector for gene therapy; however, it has not yet been used in the context of Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy
930. Muscle Stem Cells Deliver Dystrophin and Adopt a Cardiac Phenotype through Both Differentiation and Fusion in the Dystrophic (mdx) Heart Thomas R. Payne,1 Hideki Oshima,2 Tetsuro Sakai,3 Yiqun Ling,2 Burhan Gharabeih,5 James H. Cummins,5 Johnny Huard.1,4,5 1 Bioengineering; 2Cardiothoracic Surgery; 3Anesthesiology; 4 Molecular Genetic and Biochemistry; 5Orthopaedic Surgery, University of Pittsburgh, Children’s Hospital of Pittsburgh, Pittsburgh, PA. A reduction or absence of dystrophin protein in the heart will cause cardiac muscle degeneration and the progressive development of cardiomyopathy. In this study, we investigated the ability of a population of murine skeletal muscle–derived stem cells (MDSCs) to deliver dystrophin and acquire a cardiac phenotype after transplantation into the dystrophic (mdx) murine heart. The injected MDSCs engrafted and partially restored dystrophin expression within the dystrophic (mdx) myocardium for up to 12 weeks after implantation. In these hearts, approximately 3-5% of the donorlabeled cells expressed a cardiac phenotype, and more than half of these cells displayed a hybrid cardiac and skeletal muscle phenotype at 2, 4, 8 and 12 weeks after implantation. The majority of the donor cells expressing a cardiac phenotype were located along the periphery of the graft or within the host myocardium. Transplantation of female MDSCs into male recipient hearts revealed that the injected cells acquired a cardiac phenotype through both differentiation and fusion with host cardiomyocytes. The formation S356
of fibrotic tissue within the injection site may have inhibited further acquisition of a cardiac phenotype by the implanted MDSCs. The results presented here support the potential use of MDSCs to advance muscle cell–based therapies for cardiac repair.
931. Enhanced Cardiac Muscle Contractility Achieved by Engineered Zinc Finger Transcriptional Repressors of Phospholamban H. Steven Zhang,1 Lei Zhang,1 Yuxin Liang,1 Reed Hickey,2 Dmitry Guschin,1 Simon Chandler,1 Mike Kunis,1 Linda Hinh,1 Dengfeng Xia,1 Xiaohong Zhong,1 S. Kaye Spratt,1 J. Tyler Martin,1 Casey C. Case,1 Frank J. Giordano,2 Philip D. Gregory,1 Edward J. Rebar.1 1 Sangamo BioSciences, Richmond, CA; 2Yale University, School of Medicine, New Haven, CT. Phospholamban (PLN) is a critical regulator of cardiac calcium homeostasis and muscle contractility. Recent studies have demonstrated that ablation or inhibition of PLN function can improve cardiac contractile properties in animal models of congestive heart failure (CHF), suggesting that PLN is an attractive target for treatment of CHF. Because of its use of a large, relatively flat interaction surface when docking with its regulation target (Sarcoplasmic Reticulum Ca2+ ATPase 2 isoform A (SERCA2a)), the development of small-molecule inhibitors of PLN function is expected to be extremely challenging. Therefore, approaches that aim to block the expression of PLN may provide a superior means of achieving the desired therapeutic effect. As part of a therapeutic program in CHF we are developing designed zinc finger protein (ZFP) transcriptional repressors of PLN. We have engineered ZFP repressors that target either the human or rat PLN promoter. The rat-specific ZFP repressor gave >90% reduction of PLN mRNA level in a rat heart-derived cell line; and the ZFP repressor targeted to the human PLN promoter produced a similar level of repression in human smooth muscle cells. Microarray analyses indicated that both the rat- and humantargeted ZFPs operated with unrivaled specificity, with PLN being the only gene that was significantly repressed within the monitored genome (14,000-16,000 transcripts). Furthermore, when the rat PLN repressor was introduced into primary cardiomyocytes of day 1 neonates, it efficiently repressed PLN transcription, despite the high level of PLN expression in these cells. Most importantly, when the same ZFP was introduced into adult rat hearts, subsequently isolated ZFP-positive myocytes showed accelerated calcium transients as well as improved contractility, highlighting the functional significance of ZFP mediated PLN repression. Our results demonstrate that, by repressing PLN transcription, engineered ZFPs can enhance the contractile properties of cardiac myocytes, and therefore have the potential of improving cardiac function in CHF patients. The ability to design therapeutically relevant ZFPs can be applied to alter the transcription programs of many disease-related genes. Furthermore, the highly effective and specific ZFP-based approaches provide an alternative to technologies such as small interfering RNA (siRNA), and offer versatility that is unmatched by cDNA-based gene therapies.
932. SV40 Pseudovirions Are Efficient and Safe Myocardial Gene Therapy Vectors Ronen Beeri,1 Thea Pugatsch,1 Suzane Abedat,1 Mahmud Abdel Latif,2 Ariella Oppenheim.2 1 Heart Institute, Hadassah-Hebrew University Medical Center, Jerusalem, Israel; 2Hematology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel. Background: Simian virus-40 (SV40) is an efficient vector for gene therapy; however, it has not yet been used in the context of Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy