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1
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Dai L, Du L. Genes in pediatric pulmonary arterial hypertension and the most promising BMPR2 gene therapy. Front Genet 2022; 13:961848. [PMID: 36506323 PMCID: PMC9730536 DOI: 10.3389/fgene.2022.961848] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2022] [Accepted: 11/03/2022] [Indexed: 11/25/2022] Open
Abstract
Pulmonary arterial hypertension (PAH) is a rare but progressive and lethal vascular disease of diverse etiologies, mainly caused by proliferation of endothelial cells, smooth muscle cells in the pulmonary artery, and fibroblasts, which ultimately leads to right-heart hypertrophy and cardiac failure. Recent genetic studies of childhood-onset PAH report that there is a greater genetic burden in children than in adults. Since the first-identified pathogenic gene of PAH, BMPR2, which encodes bone morphogenetic protein receptor 2, a receptor in the transforming growth factor-β superfamily, was discovered, novel causal genes have been identified and substantially sharpened our insights into the molecular genetics of childhood-onset PAH. Currently, some newly identified deleterious genetic variants in additional genes implicated in childhood-onset PAH, such as potassium channels (KCNK3) and transcription factors (TBX4 and SOX17), have been reported and have greatly updated our understanding of the disease mechanism. In this review, we summarized and discussed the advances of genetic variants underlying childhood-onset PAH susceptibility and potential mechanism, and the most promising BMPR2 gene therapy and gene delivery approaches to treat childhood-onset PAH in the future.
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2
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Bisserier M, Sun XQ, Fazal S, Turnbull IC, Bonnet S, Hadri L. Novel Insights into the Therapeutic Potential of Lung-Targeted Gene Transfer in the Most Common Respiratory Diseases. Cells 2022; 11:984. [PMID: 35326434 PMCID: PMC8947048 DOI: 10.3390/cells11060984] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2022] [Revised: 03/08/2022] [Accepted: 03/10/2022] [Indexed: 12/10/2022] Open
Abstract
Over the past decades, a better understanding of the genetic and molecular alterations underlying several respiratory diseases has encouraged the development of new therapeutic strategies. Gene therapy offers new therapeutic alternatives for inherited and acquired diseases by delivering exogenous genetic materials into cells or tissues to restore physiological protein expression and/or activity. In this review, we review (1) different types of viral and non-viral vectors as well as gene-editing techniques; and (2) the application of gene therapy for the treatment of respiratory diseases and disorders, including pulmonary arterial hypertension, idiopathic pulmonary fibrosis, cystic fibrosis, asthma, alpha-1 antitrypsin deficiency, chronic obstructive pulmonary disease, non-small-cell lung cancer, and COVID-19. Further, we also provide specific examples of lung-targeted therapies and discuss the major limitations of gene therapy.
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Affiliation(s)
- Malik Bisserier
- Cardiovascular Research Institute, Icahn School of Medicine at Mount Sinai, 1470 Madison Avenue, New York, NY 10029, USA; (M.B.); (S.F.); (I.C.T.)
| | - Xiao-Qing Sun
- Department of Pulmonary Medicine, Amsterdam Cardiovascular Sciences, Amsterdam UMC, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands;
| | - Shahood Fazal
- Cardiovascular Research Institute, Icahn School of Medicine at Mount Sinai, 1470 Madison Avenue, New York, NY 10029, USA; (M.B.); (S.F.); (I.C.T.)
| | - Irene C. Turnbull
- Cardiovascular Research Institute, Icahn School of Medicine at Mount Sinai, 1470 Madison Avenue, New York, NY 10029, USA; (M.B.); (S.F.); (I.C.T.)
| | - Sébastien Bonnet
- Pulmonary Hypertension Research Group, Québec Heart and Lung Institute Research Centre, Québec, QC G1V4G5, Canada;
- Department of Medicine, Laval University, Québec, QC G1V4G5, Canada
| | - Lahouaria Hadri
- Cardiovascular Research Institute, Icahn School of Medicine at Mount Sinai, 1470 Madison Avenue, New York, NY 10029, USA; (M.B.); (S.F.); (I.C.T.)
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3
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Fujino K, Yamamoto Y, Daito T, Makino A, Honda T, Tomonaga K. Generation of a non-transmissive Borna disease virus vector lacking both matrix and glycoprotein genes. Microbiol Immunol 2018; 61:380-386. [PMID: 28776750 DOI: 10.1111/1348-0421.12505] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2017] [Revised: 07/24/2017] [Accepted: 08/01/2017] [Indexed: 11/30/2022]
Abstract
Borna disease virus (BoDV), a prototype of mammalian bornavirus, is a non-segmented, negative strand RNA virus that often causes severe neurological disorders in infected animals, including horses and sheep. Unique among animal RNA viruses, BoDV transcribes and replicates non-cytopathically in the cell nucleus, leading to establishment of long-lasting persistent infection. This striking feature of BoDV indicates its potential as an RNA virus vector system. It has previously been demonstrated by our team that recombinant BoDV (rBoDV) lacking an envelope glycoprotein (G) gene develops persistent infections in transduced cells without loss of the viral genome. In this study, a novel non-transmissive rBoDV, rBoDV ΔMG, which lacks both matrix (M) and G genes in the genome, is reported. rBoDV-ΔMG expressing green fluorescence protein (GFP), rBoDV ΔMG-GFP, was efficiently generated in Vero/MG cells stably expressing both BoDV M and G proteins. Infection with rBoDV ΔMG-GFP was persistently maintained in the parent Vero cells without propagation within cell culture. The optimal ratio of M and G for efficient viral particle production by transient transfection of M and G expression plasmids into cells persistently infected with rBoDV ΔMG-GFP was also demonstrated. These findings indicate that the rBoDV ΔMG-based BoDV vector may provide an extremely safe virus vector system and could be a novel strategy for investigating the function of M and G proteins and the host range of bornaviruses.
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Affiliation(s)
- Kan Fujino
- Laboratory of RNA Viruses, Department of Virus Research, Institute for Frontier Life and Medical Sciences, Japan
| | - Yusuke Yamamoto
- Laboratory of RNA Viruses, Department of Virus Research, Institute for Frontier Life and Medical Sciences, Japan.,Department of Mammalian Regulatory Network, Graduate School of Biostudies, Japan
| | - Takuji Daito
- Laboratory of RNA Viruses, Department of Virus Research, Institute for Frontier Life and Medical Sciences, Japan
| | - Akiko Makino
- Laboratory of RNA Viruses, Department of Virus Research, Institute for Frontier Life and Medical Sciences, Japan
| | - Tomoyuki Honda
- Laboratory of RNA Viruses, Department of Virus Research, Institute for Frontier Life and Medical Sciences, Japan
| | - Keizo Tomonaga
- Laboratory of RNA Viruses, Department of Virus Research, Institute for Frontier Life and Medical Sciences, Japan.,Department of Mammalian Regulatory Network, Graduate School of Biostudies, Japan.,Department of Molecular Virology, Graduate School of Medicine, Kyoto University, 53 Kawahara-cho Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
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4
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Schott JW, Morgan M, Galla M, Schambach A. Viral and Synthetic RNA Vector Technologies and Applications. Mol Ther 2016; 24:1513-27. [PMID: 27377044 DOI: 10.1038/mt.2016.143] [Citation(s) in RCA: 56] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2015] [Accepted: 06/30/2016] [Indexed: 12/21/2022] Open
Abstract
Use of RNA is an increasingly popular method to transiently deliver genetic information for cell manipulation in basic research and clinical therapy. In these settings, viral and nonviral RNA platforms are employed for delivery of small interfering RNA and protein-coding mRNA. Technological advances allowing RNA modification for increased stability, improved translation and reduced immunogenicity have led to increased use of nonviral synthetic RNA, which is delivered in naked form or upon formulation. Alternatively, highly efficient viral entry pathways are exploited to transfer genes of interest as RNA incorporated into viral particles. Current viral RNA transfer technologies are derived from Retroviruses, nonsegmented negative-strand RNA viruses or positive-stranded Alpha- and Flaviviruses. In retroviral particles, the genes of interest can either be incorporated directly into the viral RNA genome or as nonviral RNA. Nonsegmented negative-strand virus-, Alpha- and Flavivirus-derived vectors support prolonged expression windows through replication of viral RNA encoding genes of interest. Mixed technologies combining viral and nonviral components are also available. RNA transfer is ideal for all settings that do not require permanent transgene expression and excludes potentially detrimental DNA integration into the target cell genome. Thus, RNA-based technologies are successfully applied for reprogramming, transdifferentiation, gene editing, vaccination, tumor therapy, and gene therapy.
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Affiliation(s)
- Juliane W Schott
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany
| | - Michael Morgan
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany
| | - Melanie Galla
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany
| | - Axel Schambach
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany.,Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA
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5
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Baron J, Baron MD. Development of a helper cell-dependent form of peste des petits ruminants virus: a system for making biosafe antigen. Vet Res 2015; 46:101. [PMID: 26396073 PMCID: PMC4579661 DOI: 10.1186/s13567-015-0231-y] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2015] [Accepted: 07/29/2015] [Indexed: 11/10/2022] Open
Abstract
Peste des petits ruminants (PPR) is a viral disease of sheep and goats that is spreading through many countries in the developing world. Work on the virus is often restricted to studies of attenuated vaccine strains or to work in laboratories that have high containment facilities. We have created a helper cell dependent form of PPR virus by removing the entire RNA polymerase gene and complementing it with polymerase made constitutively in a cell line. The resultant L-deleted virus grows efficiently in the L-expressing cell line but not in other cells. Virus made with this system is indistinguishable from normal virus when used in diagnostic assays, and can be grown in normal facilities without the need for high level biocontainment. The L-deleted virus will thus make a positive contribution to the control and study of this important disease.
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Affiliation(s)
- Jana Baron
- The Pirbright Institute, Ash Road, Pirbright, Surrey, GU24 0NF, UK.
| | - Michael D Baron
- The Pirbright Institute, Ash Road, Pirbright, Surrey, GU24 0NF, UK.
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6
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Kurioka T, Mizutari K, Niwa K, Fukumori T, Inoue M, Hasegawa M, Shiotani A. Hyaluronic acid pretreatment for Sendai virus-mediated cochlear gene transfer. Gene Ther 2015; 23:187-95. [PMID: 26361273 DOI: 10.1038/gt.2015.94] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2015] [Revised: 08/19/2015] [Accepted: 09/04/2015] [Indexed: 12/14/2022]
Abstract
Gene therapy with viral vectors is one of the most promising strategies for sensorineural hearing loss. However, safe and effective administration of the viral vector into cochlear tissue is difficult because of the anatomical isolation of the cochlea. We investigated the efficiency and safety of round window membrane (RWM) application of Sendai virus, one of the most promising non-genotoxic vectors, after pretreatment with hyaluronic acid (HA) on the RWM to promote efficient viral translocation into the cochlea. Sendai virus expressing the green fluorescent protein reporter gene was detected throughout cochlear tissues following application combined with HA pretreatment. Quantitative analysis revealed that maximum expression was reached 3 days after treatment. The efficiency of transgene expression was several 100-fold greater with HA pretreatment than that without. Furthermore, unlike the conventional intracochlear delivery methods, this approach did not cause hearing loss. These findings reveal the potential utility of gene therapy with Sendai virus and HA for treatment of sensorineural hearing loss.
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Affiliation(s)
- T Kurioka
- Department of Otolaryngology, Head and Neck Surgery, National Defense Medical College, Saitama, Japan
| | - K Mizutari
- Department of Otolaryngology, Head and Neck Surgery, National Defense Medical College, Saitama, Japan
| | - K Niwa
- Department of Otolaryngology, Head and Neck Surgery, National Defense Medical College, Saitama, Japan
| | - T Fukumori
- Department of Otolaryngology, Head and Neck Surgery, National Defense Medical College, Saitama, Japan
| | - M Inoue
- DNAVEC Corporation, Ibaraki, Japan
| | | | - A Shiotani
- Department of Otolaryngology, Head and Neck Surgery, National Defense Medical College, Saitama, Japan
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7
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Matsushita K, Shimada H, Ueda Y, Inoue M, Hasegawa M, Tomonaga T, Matsubara H, Nomura F. Non-transmissible Sendai virus vector encoding c-myc suppressor FBP-interacting repressor for cancer therapy. World J Gastroenterol 2014; 20:4316-4328. [PMID: 24764668 PMCID: PMC3989966 DOI: 10.3748/wjg.v20.i15.4316] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/22/2013] [Revised: 12/14/2013] [Accepted: 01/20/2014] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element (FUSE)-binding protein-interacting repressor (FIR).
METHODS: Endogenous c-Myc suppression and apoptosis induction by a transient FIR-expressing vector was examined in vivo via a HA-tagged FIR (HA-FIR) expression vector. A fusion gene-deficient, non-transmissible, Sendai virus (SeV) vector encoding FIR cDNA, SeV/dF/FIR, was prepared. SeV/dF/FIR was examined for its gene transduction efficiency, viral dose dependency of antitumor effect and apoptosis induction in HeLa (cervical squamous cell carcinoma) cells and SW480 (colon adenocarcinoma) cells. Antitumor efficacy in a mouse xenograft model was also examined. The molecular mechanism of the anti-tumor effect and c-Myc suppression by SeV/dF/FIR was examined using Spliceostatin A (SSA), a SAP155 inhibitor, or SAP155 siRNA which induce c-Myc by increasing FIR∆exon2 in HeLa cells.
RESULTS: FIR was found to repress c-myc transcription and in turn the overexpression of FIR drove apoptosis through c-myc suppression. Thus, FIR expressing vectors are potentially applicable for cancer therapy. FIR is alternatively spliced by SAP155 in cancer cells lacking the transcriptional repression domain within exon 2 (FIR∆exon2), counteracting FIR for c-Myc protein expression. Furthermore, FIR forms a complex with SAP155 and inhibits mutual well-established functions. Thus, both the valuable effects and side effects of exogenous FIR stimuli should be tested for future clinical application. SeV/dF/FIR, a cytoplasmic RNA virus, was successfully prepared and showed highly efficient gene transduction in in vivo experiments. Furthermore, in nude mouse tumor xenograft models, SeV/dF/FIR displayed high antitumor efficiency against human cancer cells. SeV/dF/FIR suppressed SSA-activated c-Myc. SAP155 siRNA, potentially produces FIR∆exon2, and led to c-Myc overexpression with phosphorylation at Ser62. HA-FIR suppressed endogenous c-Myc expression and induced apoptosis in HeLa and SW480 cells. A c-myc transcriptional suppressor FIR expressing SeV/dF/FIR showed high gene transduction efficiency with significant antitumor effects and apoptosis induction in HeLa and SW480 cells.
CONCLUSION: SeV/dF/FIR showed strong tumor growth suppression with no significant side effects in an animal xenograft model, thus SeV/dF/FIR is potentially applicable for future clinical cancer treatment.
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8
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Hara H, Mouri A, Yonemitsu Y, Nabeshima T, Tabira T. Mucosal immunotherapy in an Alzheimer mouse model by recombinant Sendai virus vector carrying Aβ1-43/IL-10 cDNA. Vaccine 2011; 29:7474-82. [PMID: 21803105 DOI: 10.1016/j.vaccine.2011.07.057] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2011] [Revised: 06/28/2011] [Accepted: 07/17/2011] [Indexed: 11/18/2022]
Abstract
Based on the amyloid cascade hypothesis, many reports have indicated that immunotherapy is beneficial for Alzheimer's disease (AD). We developed a mucosal immunotherapy for AD by nasal administration of recombinant Sendai virus vector carrying Aβ1-43 and mouse IL-10 cDNA. Nasal but not intramuscular administration of the vaccine induced good antibody responses to Aβ. When APP transgenic mice (Tg2576) received this vaccine once nasally, the Aβ plaque burden was significantly decreased 8 weeks after without inducing inflammation in the brain. The amount of Aβ measured by ELISA was also reduced in both soluble and insoluble fractions of the brain homogenates, and notably the Aβ oligomer (12-mer) was also apparently decreased. Tg2576 mice showed significant improvement in cognitive functions examined at 3 months after vaccination. Thus, this is an alternative immunotherapy for AD, which has an advantage in non-invasive, safe and relatively long lasting features.
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Affiliation(s)
- Hideo Hara
- Division of Neurology, Department of Internal Medicine, Saga University Faculty of Medicine, 5-1-1 Nabeshima, Saga 849-8501, Japan
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9
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A respiratory syncytial virus replicon that is noncytotoxic and capable of long-term foreign gene expression. J Virol 2011; 85:4792-801. [PMID: 21389127 DOI: 10.1128/jvi.02399-10] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
Respiratory syncytial virus (RSV) infection of most cultured cell lines causes cell-cell fusion and death. Cell fusion is caused by the fusion (F) glycoprotein and is clearly cytopathic, but other aspects of RSV infection may also contribute to cytopathology. To investigate this possibility, we generated an RSV replicon that lacks all three of its glycoprotein genes and so cannot cause cell-cell fusion or virus spread. This replicon includes a green fluorescent protein gene and an antibiotic resistance gene to enable detection and selection of replicon-containing cells. Adaptive mutations in the RSV replicon were not required for replicon maintenance. Cells containing the replicon could be cloned and passaged many times in the absence of antibiotic selection, with 99% or more of the cells retaining the replicon after each cell division. Transient expression of the F and G (attachment) glycoproteins supported the production of virions that could transfer the replicon into most cell lines tested. Since the RSV replicon is not toxic to these cultured cells and does not affect their rate of cell division, none of the 8 internal viral proteins, the viral RNA transcripts, or the host response to these molecules or their activities is cytopathic. However, the level of replicon genome and gene expression is controlled in some manner well below that of complete virus and, as such, might avoid cytotoxicity. RSV replicons could be useful for cytoplasmic gene expression in vitro and in vivo and for screening for compounds active against the viral polymerase.
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10
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Zimmer G. RNA replicons - a new approach for influenza virus immunoprophylaxis. Viruses 2010; 2:413-434. [PMID: 21994644 PMCID: PMC3185613 DOI: 10.3390/v2020413] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2009] [Revised: 01/25/2010] [Accepted: 01/27/2010] [Indexed: 02/07/2023] Open
Abstract
RNA replicons are derived from either positive- or negative-strand RNA viruses. They represent disabled virus vectors that are not only avirulent, but also unable to revert to virulence. Due to autonomous RNA replication, RNA replicons are able to drive high level, cytosolic expression of recombinant antigens stimulating both the humoral and the cellular branch of the immune system. This review provides an update on the available literature covering influenza virus vaccines based on RNA replicons. The pros and cons of these vaccine strategies will be discussed and future perspectives disclosed.
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Affiliation(s)
- Gert Zimmer
- Institute of Virology and Immunoprophylaxis (IVI), Sensemattstrasse 293, CH-3147 Mittelhäusern, Switzerland
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11
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Del Bigio MR. Ependymal cells: biology and pathology. Acta Neuropathol 2010; 119:55-73. [PMID: 20024659 DOI: 10.1007/s00401-009-0624-y] [Citation(s) in RCA: 257] [Impact Index Per Article: 17.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2009] [Revised: 12/03/2009] [Accepted: 12/04/2009] [Indexed: 11/28/2022]
Abstract
The literature was reviewed to summarize the current understanding of the role of ciliated ependymal cells in the mammalian brain. Previous reviews were summarized. Publications from the past 10 years highlight interactions between ependymal cells and the subventricular zone and the possible role of restricted ependymal populations in neurogenesis. Ependymal cells provide trophic support and possibly metabolic support for progenitor cells. Channel proteins such as aquaporins may be important for determining water fluxes at the ventricle wall. The junctional and anchoring proteins are now fairly well understood, as are proteins related to cilia function. Defects in ependymal adhesion and cilia function can cause hydrocephalus through several different mechanisms, one possibility being loss of patency of the cerebral aqueduct. Ependymal cells are susceptible to infection by a wide range of common viruses; while they may act as a line of first defense, they eventually succumb to repeated attacks in long-lived organisms. Ciliated ependymal cells are almost certainly important during brain development. However, the widespread absence of ependymal cells from the adult human lateral ventricles suggests that they may have only regionally restricted value in the mature brain of large size.
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Affiliation(s)
- Marc R Del Bigio
- Department of Pathology, University of Manitoba, Winnipeg, MB, Canada.
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12
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Huang J, Inoue M, Hasegawa M, Tomihara K, Tanaka T, Chen J, Hamada H. Sendai viral vector mediated angiopoietin-1 gene transfer for experimental ischemic limb disease. Angiogenesis 2009; 12:243-9. [PMID: 19322669 DOI: 10.1007/s10456-009-9144-6] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2009] [Accepted: 03/13/2009] [Indexed: 11/27/2022]
Abstract
Sendai virus vector is emerging as a promising vector for gene therapy, and angiopoietin-1 (Ang-1) has been reported to improve the blood flow recovery in the ischemic limb or heart. In this study, we constructed a human Ang-1-expressing Sendai viral vector (SeVhAng-1) and injected it into the ischemic limb of rats. We found that SeVhAng-1 improved the blood flow recovery and increased the capillary density of the ischemic limb, compared with the controls. We also found that SeVhAng-1 increased p-Akt during the early period of limb ischemia, and decreased apoptosis in ischemic limb. It suggests that SeVhAng-1 may serve as a potential therapeutic tool in ischemic limb disease.
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Affiliation(s)
- Jianhua Huang
- Department of Molecular Medicine, Sapporo Medical University, Chuo-ku, Sapporo, Japan.
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13
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Abstract
Dendritic cells (DCs) have a crucial role to play in fighting nonself organisms and cells, including tumors. Clinically, numerous DC vaccinations have been attempted for cancer immunotherapy since the first trial, published in 1995, but with limited success. We found that Sendai virus (SeV) vector infection induces maturation of DCs and produces more powerful antitumor immunity against DCs in mouse models. We used a SeV vector as an immune booster for tumors and believe that this novel therapy, designated as "immunostimulatory virotherapy," will offer potent treatment for tumors.
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Affiliation(s)
- Yasuji Ueda
- Department of Gene Therapy, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba, 260-8670, Japan.
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14
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Generation of optimized and urokinase-targeted oncolytic Sendai virus vectors applicable for various human malignancies. Gene Ther 2008; 16:392-403. [PMID: 19037241 DOI: 10.1038/gt.2008.167] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
We previously reported the development of a prototype 'oncolytic Sendai virus (SeV) vector' formed by introducing two major genomic modifications to the original SeV, namely deletion of the matrix (M) gene to avoid budding of secondary viral particles and manipulation of the trypsin-dependent cleavage site of the fusion (F) gene to generate protease-specific sequences. As a result, the 'oncolytic SeV' that was susceptible to matrix metalloproteinases (MMPs) was shown to selectively kill MMP-expressing tumors through syncytium formation in vitro and in vivo. However, its efficacy has been relatively limited because of the requirement of higher expression of MMPs and smaller populations of MMP-expressing tumors. To overcome these limitations, we have designed an optimized and dramatically powerful oncolytic SeV vector. Truncation of 14-amino acid residues of the cytoplasmic domain of F protein resulted in dramatic enhancement of cell-killing activities of oncolytic SeV, and the combination with replacement of the trypsin cleavage site with the new urokinase type plasminogen activator (uPA)-sensitive sequence (SGRS) led a variety of human tumors, including prostate (PC-3), renal (CAKI-I), pancreatic (BxPC3) and lung (PC14) cancers, to extensive death through massive cell-to-cell spreading without significant dissemination to the surrounding noncancerous tissue in vivo. These results indicate a dramatic improvement of antitumor activity; therefore, extensive utility of the newly designed uPA-targeted oncolytic SeV has significant potential for treating patients bearing urokinase-expressing cancers in clinical settings.
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15
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Jin G, Inoue M, Hayashi T, Deguchi K, Nagotani S, Zhang H, Wang X, Shoji M, Hasegawa M, Abe K. Sendai virus-mediated gene transfer of GDNF reduces AIF translocation and ameliorates ischemic cerebral injury. Neurol Res 2008; 30:731-9. [PMID: 18593521 DOI: 10.1179/174313208x305418] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
The therapeutic effect of a novel RNA viral vector, Sendai virus (SeV)-mediated glial cell line-derived neurotrophic factor (GDNF) gene (SeV/GDNF), on the infarct volume, was investigated after 90 minutes of transient middle cerebral artery occlusion (tMCAO) in rats with relation to nuclear translocation of apoptosis inducing factor (AIF). The topical administration of SeV/GDNF induced high level expression of GDNF protein, which effectively reduced the infarct volume when administrated 0 and 1 hours as well after the reperfusion. Twenty-four hours after ischemia, the obvious nuclear translocation of AIF was found in neurons of peri-infarct area, which significantly reduced with administration of SeV/GDNF 0 or 1 hour after reperfusion, as well as the number of TUNEL positive cells. These results demonstrate that SeV vector-mediated gene transfer of GDNF effectively reduced ischemic infarct volume after tMCAO and extended the therapeutic time window compared with previous viral vectors, and that promoting neuronal survival of GDNF might be related to the reduction of AIF nuclear translocation, indicating the high therapeutic potency of SeV/GDNF for cerebral ischemia.
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Affiliation(s)
- Guang Jin
- Department of Neurology, Graduate School of Medicine and Dentistry, Okayama University, Okayama, Japan.
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16
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Ban H, Inoue M, Griesenbach U, Munkonge F, Chan M, Iida A, Alton EWFW, Hasegawa M. Expression and maturation of Sendai virus vector-derived CFTR protein: functional and biochemical evidence using a GFP-CFTR fusion protein. Gene Ther 2007; 14:1688-94. [PMID: 17898794 DOI: 10.1038/sj.gt.3303032] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
Sendai virus (SeV) vector has been shown to efficiently transduce airway epithelial cells. As a precursor to the potential use of this vector for cystic fibrosis (CF) gene therapy, the correct maturation of the SeV vector-derived CF transmembrane conductance regulator (CFTR) protein was examined using biochemical and functional analyses. We constructed a recombinant SeV vector, based on the fusion (F) gene-deleted non-transmissible SeV vector, carrying the GFP-CFTR gene in which the N terminus of CFTR was fused to green fluorescence protein (GFP). This vector was recovered and propagated to high titers in the packaging cell line. Western blotting using an anti-GFP antibody detected both the fully glycosylated (mature) and the core-glycosylated (immature) proteins, indicating that SeV vector-derived GFP-CFTR was similar to endogenous CFTR. We also confirmed the functional channel activity of GFP-CFTR in an iodide efflux assay. The efficient expression of GFP-CFTR, and its apical surface localization, were observed in both MDCK cells in vitro, and in the nasal epithelium of mice in vivo. We concluded that recombinant SeV vector, a cytoplasmically maintained RNA vector, is able to direct production of a correctly localized, mature form of CFTR, suggesting the value of this vector for studies of CF gene therapy.
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Affiliation(s)
- H Ban
- DNAVEC Corporation, Tsukuba-shi, Ibaraki, Japan
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17
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Nishimura K, Segawa H, Goto T, Morishita M, Masago A, Takahashi H, Ohmiya Y, Sakaguchi T, Asada M, Imamura T, Shimotono K, Takayama K, Yoshida T, Nakanishi M. Persistent and stable gene expression by a cytoplasmic RNA replicon based on a noncytopathic variant Sendai virus. J Biol Chem 2007; 282:27383-27391. [PMID: 17623660 DOI: 10.1074/jbc.m702028200] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Persistent and stable expression of foreign genes has been achieved in mammalian cells by integrating the genes into the host chromosomes. However, this approach has several shortcomings in practical applications. For example, large scale production of protein pharmaceutics frequently requires laborious amplification of the inserted genes to optimize the gene expression. The random chromosomal insertion of exogenous DNA also results occasionally in malignant transformation of normal tissue cells, raising safety concerns in medical applications. Here we report a novel cytoplasmic RNA replicon capable of expressing installed genes stably without chromosome insertion. This system is based on the RNA genome of a noncytopathic variant Sendai virus strain, Cl.151. We found that this variant virus establishes stable symbiosis with host cells by escaping from retinoic acid-inducible gene I-interferon regulatory factor 3-mediated antiviral machinery. Using a cloned genome cDNA of Sendai virus Cl.151, we developed a recombinant RNA installed with exogenous marker genes that was maintained stably in the cytoplasm as a high copy replicon (about 4 x 10(4) copies/cell) without interfering with normal cellular function. Strong expression of the marker genes persisted for more than 6 months in various types of cultured cells and for at least two months in rat colonic mucosa without any apparent side effects. This stable RNA replicon is a potentially valuable genetic platform for various biological applications.
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Affiliation(s)
- Ken Nishimura
- Biotherapeutic Research Laboratory and the National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan; Japan Society for Promotion of Science, 6 Ichibancho, Chiyoda-ku, Tokyo 102-8471, Japan, the
| | - Hiroaki Segawa
- Biotherapeutic Research Laboratory and the National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan
| | - Takahiro Goto
- Department of Pharmaceutics, Hoshi University, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan, the
| | - Mariko Morishita
- Department of Pharmaceutics, Hoshi University, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan, the
| | - Akinori Masago
- Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan, the
| | - Hitoshi Takahashi
- Department of Viral Oncology, Institute for Virus Research, Kyoto University, 53 Kawahara-cho, Shogo-in, Sakyo-ku, Kyoto 606-8507, Japan, the
| | - Yoshihiro Ohmiya
- Resarch Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan, and the
| | - Takemasa Sakaguchi
- Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan
| | - Masahiro Asada
- Signaling Molecules Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan, the
| | - Toru Imamura
- Signaling Molecules Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan, the
| | - Kunitada Shimotono
- Department of Viral Oncology, Institute for Virus Research, Kyoto University, 53 Kawahara-cho, Shogo-in, Sakyo-ku, Kyoto 606-8507, Japan, the
| | - Kozo Takayama
- Department of Pharmaceutics, Hoshi University, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan, the
| | - Tetsuya Yoshida
- Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan
| | - Mahito Nakanishi
- Biotherapeutic Research Laboratory and the National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan.
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18
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Ferrari S, Griesenbach U, Iida A, Farley R, Wright AM, Zhu J, Munkonge FM, Smith SN, You J, Ban H, Inoue M, Chan M, Singh C, Verdon B, Argent BE, Wainwright B, Jeffery PK, Geddes DM, Porteous DJ, Hyde SC, Gray MA, Hasegawa M, Alton EWFW. Sendai virus-mediated CFTR gene transfer to the airway epithelium. Gene Ther 2007; 14:1371-9. [PMID: 17597790 DOI: 10.1038/sj.gt.3302991] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The potential for gene therapy to be an effective treatment for cystic fibrosis has been hampered by the limited gene transfer efficiency of current vectors. We have shown that recombinant Sendai virus (SeV) is highly efficient in mediating gene transfer to differentiated airway epithelial cells, because of its capacity to overcome the intra- and extracellular barriers known to limit gene delivery. Here, we have identified a novel method to allow the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA sequence to be inserted within SeV (SeV-CFTR). Following in vitro transduction with SeV-CFTR, a chloride-selective current was observed using whole-cell and single-channel patch-clamp techniques. SeV-CFTR administration to the nasal epithelium of cystic fibrosis (CF) mice (Cftr(G551D) and Cftr(tm1Unc)TgN(FABPCFTR)#Jaw mice) led to partial correction of the CF chloride transport defect. In addition, when compared to a SeV control vector, a higher degree of inflammation and epithelial damage was found in the nasal epithelium of mice treated with SeV-CFTR. Second-generation transmission-incompetent F-deleted SeV-CFTR led to similar correction of the CF chloride transport defect in vivo as first-generation transmission-competent vectors. Further modifications to the vector or the host may make it easier to translate these studies into clinical trials of cystic fibrosis.
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Affiliation(s)
- S Ferrari
- Department of Gene Therapy, Faculty of Medicine, Imperial College, National Heart and Lung Institute, London, UK
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19
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Kanzaki S, Shiotani A, Inoue M, Hasegawa M, Ogawa K. Sendai Virus Vector-Mediated Transgene Expression in the Cochlea in vivo. ACTA ACUST UNITED AC 2007; 12:119-26. [PMID: 17264475 DOI: 10.1159/000097798] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2005] [Accepted: 09/15/2006] [Indexed: 11/19/2022]
Abstract
We injected a recombinant Sendai virus (SeV) vector into the guinea pig cochlea using two different approaches--the scala media and scala tympani--and investigated which cell types took up the vector. The hearing threshold shift and distribution of transfected cells in animals using the scala media approach were different compared to those using the scala tympani approach. SeV can transfect very different types of cells, including stria vascularis, spiral ganglion neurons, and sensory epithelia of the organ of Corti, and fibrocytes of the scala tympani. Because SeV vectors can potentially deliver stimuli to the cochlea to induce hair cell regeneration, it may be a powerful tool for repairing the organ of Corti.
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Affiliation(s)
- Sho Kanzaki
- Department of Otolaryngology, Keio University, Tokyo, Japan.
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20
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Fujita S, Eguchi A, Okabe J, Harada A, Sasaki K, Ogiwara N, Inoue Y, Ito T, Matsuda H, Kataoka K, Kato A, Hasegawa M, Nakanishi M. Sendai virus-mediated gene delivery into hepatocytes via isolated hepatic perfusion. Biol Pharm Bull 2006; 29:1728-34. [PMID: 16880633 DOI: 10.1248/bpb.29.1728] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The recombinant Sendai virus vector is a promising tool for human gene therapy, capable of inducing high-level expression of therapeutic genes in tissue cells in situ. The target tissues include airway epithelium, blood vessels, skeletal muscle, retina and the central nervous system, but application to hepatic tissues has not yet been achieved, because direct intraportal injection of the vector is not feasible. We report an efficient and harmless procedure of gene delivery by recombinant Sendai virus into rat parenchymal hepatocytes, based on isolated hepatic perfusion with controlled inflow. Critical parameters for successful hepatic gene delivery are a brief preperfusion period (25 degrees C, 5 min); appropriate vector concentration in the perfusate (10(7) pfu/ml); moderate portal vein pressure (12 mmHg) and a brief hyperthermic postperfusion period (42 degrees C, 5 min). Under these optimized conditions, marker genes were expressed in most parenchymal hepatocytes without significant damage to hepatic tissues. Furthermore, expression of the marker genes was undetectable in nonhepatic tissues, including the gonads, indicating that this approach strictly targets hepatic tissues and thus offers good clinical potential for human gene therapy.
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Affiliation(s)
- Shigeo Fujita
- Department of Surgery, E1, Osaka University Graduate School of Medicine, Japan
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21
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Griesenbach U, Boyton RJ, Somerton L, Garcia SE, Ferrari S, Owaki T, Ya-Fen Z, Geddes DM, Hasegawa M, Altmann DM, Alton EWFW. Effect of tolerance induction to immunodominant T-cell epitopes of Sendai virus on gene expression following repeat administration to lung. Gene Ther 2006; 13:449-56. [PMID: 16319950 DOI: 10.1038/sj.gt.3302677] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2005] [Revised: 09/29/2005] [Accepted: 10/01/2005] [Indexed: 11/09/2022]
Abstract
Sendai virus (SeV) is able to transfect airway epithelial cells efficiently in vivo. However, as with other viral vectors, repeated administration leads to reduced gene expression. We have investigated the impact of inducing immunological tolerance to immunodominant T-cell epitopes on gene expression following repeated administration. Immunodominant CD4 and CD8 T-cell peptide epitopes of SeV were administered to C57BL/6 mice intranasally 10 days before the first virus administration with transmission-incompetent F-protein-deleted DeltaF/SeV-GFP. At 21 days after the first virus administration, mice were again transfected with DeltaF/SeV. To avoid interference of anti-GFP antibodies, the second transfection was carried out with DeltaF/SeV-lacZ. At 2 days after the final transfection lung beta-galactosidase expression, T-cell proliferation and antibody responses were measured. A state of 'split tolerance' was achieved with reduced T-cell proliferation, but no impact on antiviral antibody production. There was no enhancement of expression on repeat administration; instead, T-cell tolerance was, paradoxically, associated with a more profound extinction of viral expression. Multiple immune mechanisms operate to eradicate viruses from the lung, and these findings indicate that impeding the adaptive T-cell response to the immunodominant viral epitope is not sufficient to prevent the process.
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Affiliation(s)
- U Griesenbach
- Department of Gene Therapy, NHLI, Imperial College, Edinburgh, London, UK.
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22
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Yoshizaki M, Hironaka T, Iwasaki H, Ban H, Tokusumi Y, Iida A, Nagai Y, Hasegawa M, Inoue M. Naked Sendai virus vector lacking all of the envelope-related genes: reduced cytopathogenicity and immunogenicity. J Gene Med 2006; 8:1151-9. [PMID: 16841365 DOI: 10.1002/jgm.938] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Sendai virus (SeV) is a new class of cytoplasmic RNA vector that is free from genotoxicity that infects and multiplies in most mammalian cells, and directs high-level transgene expression. We improved the vector by deleting all of the envelope-related genes from the SeV genome and thus reducing its immunogenicity. METHODS The matrix (M), fusion (F) and hemagglutinin-neuraminidase (HN) genes-deleted SeV vector (SeV/DeltaMDeltaFDeltaHN) was recovered in a newly established packaging cell line. Then, the generated SeV/DeltaMDeltaFDeltaHN vector was characterised by comparing with single gene-deleted type SeV vectors. RESULTS This SeV/DeltaMDeltaFDeltaHN vector carrying the green fluorescent protein gene in place of the envelope-related genes could be propagated to a titer of more than 10(8) cell infectious units/ml. This vector showed an efficient transduction capability in vitro and in vivo, and the cytopathic effect and induction of neutralizing antibody in vivo were greatly reduced compared with those of single gene-deleted type SeV vectors. No activity of neutralizing antibody or anti-HN antibody was seen when SeV/DeltaMDeltaFDeltaHN was transduced ex vivo. Additional introduction of amino acid mutations that had been identified from SeV strains causing persistent infections was also effective for the reduction of cytopathic effects. CONCLUSIONS The deletion of genes from the SeV genome and the additional mutation are very effective for reducing both the immunogenic and cytopathic reactions to the SeV vector. These modifications are expected to improve the safety and broaden the range of clinical applications of this new class of cytoplasmic RNA vector.
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Affiliation(s)
- Mariko Yoshizaki
- DNAVEC Corporation, 1-25-11 Kannondai, Tsukuba-shi, Ibaraki 305-0856, Japan
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23
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Iwadate Y, Inoue M, Saegusa T, Tokusumi Y, Kinoh H, Hasegawa M, Tagawa M, Yamaura A, Shimada H. Recombinant Sendai virus vector induces complete remission of established brain tumors through efficient interleukin-2 gene transfer in vaccinated rats. Clin Cancer Res 2005; 11:3821-7. [PMID: 15897582 DOI: 10.1158/1078-0432.ccr-04-1485] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE Sendai virus (SeV), a murine parainfluenza virus type I, replicates independent of cellular genome and directs high-level gene expressions when used as a viral vector. We constructed a nontransmissible recombinant SeV vector by deleting the matrix (M) and fusion (F) genes from its genome (SeV/DeltaMDeltaF) to enhance its safety. We also estimated the therapeutic efficacy of the novel vector system against a rat glioblastoma model. EXPERIMENTAL DESIGN We administered the recombinant SeV vector carrying the lacZ gene or the human interleukin-2 (hIL-2) gene into established 9L brain tumors in vivo simultaneous with peripheral vaccination using irradiated 9L cells. Sequential monitoring with magnetic resonance imaging was used to evaluate the therapeutic efficacy. RESULTS We found extensive transduction of the lacZ gene into the brain tumors and confirmed sufficient amounts of interleukin 2 (IL-2) production by hIL2-SeV/DeltaMDeltaF both in vitro and in vivo. The magnetic resonance imaging study showed that the intracerebral injection of hIL2-SeV/DeltaMDeltaF brought about significant reduction of the tumor growth, including complete elimination of the established brain tumors. The (51)Cr release assay showed that significant amounts of 9L-specific cytotoxic T cells were induced by the peripheral vaccination. Immunohistochemical analysis revealed that CD4(+) T cells and CD8(+) T cells were abundantly infiltrated in the target tumors. CONCLUSION The present results show that the recombinant nontransmissible SeV vector provides efficient in vivo gene transfer that induces significant regression of the established brain tumors and suggest that it will be a safe and useful viral vector for the clinical practice of glioma gene therapy.
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Affiliation(s)
- Yasuo Iwadate
- Department of Neurological Surgery, Graduate School of Medicine, Chiba University, Japan.
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24
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Sasaki K, Inoue M, Shibata H, Ueda Y, Muramatsu SI, Okada T, Hasegawa M, Ozawa K, Hanazono Y. Efficient and stable Sendai virus-mediated gene transfer into primate embryonic stem cells with pluripotency preserved. Gene Ther 2004; 12:203-10. [PMID: 15483665 DOI: 10.1038/sj.gt.3302409] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
Efficient gene transfer and regulated transgene expression in primate embryonic stem (ES) cells are highly desirable for future applications of the cells. In the present study, we have examined using the nonintegrating Sendai virus (SeV) vector to introduce the green fluorescent protein (GFP) gene into non-human primate cynomolgus ES cells. The GFP gene was vigorously and stably expressed in the cynomolgus ES cells for a year. The cells were able to form fluorescent teratomas when transplanted into immunodeficient mice. They were also able to differentiate into fluorescent embryoid bodies, neurons, and mature blood cells. In addition, the GFP expression levels were reduced dose-dependently by the addition of an anti-RNA virus drug, ribavirin, to the culture. Thus, SeV vector will be a useful tool for efficient gene transfer into primate ES cells and the method of using antiviral drugs should allow further investigation for regulated SeV-mediated gene expression.
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Affiliation(s)
- K Sasaki
- Center for Molecular Medicine, Jichi Medical School, Minamikawachi, Tochigi, Japan
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