論文 - 池田 康博
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Murakami Y., Ikeda Y., Yonemitsu Y., Miyazaki M., Inoue M., Hasegawa M., Sueishi K., Ishibashi T.
Human Gene Therapy 21 ( 2 ) 199 - 209 2010年2月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Human Gene Therapy
Lentiviral vectors are promising tools for the treatment of chronic retinal diseases, including age-related macular degeneration (AMD), as they enable stable transgene expression. On the other hand, Sendai virus (SeV) vectors provide the unique advantage of rapid gene transfer. Here we show that novel simian immunodeficiency viral vectors pseudotyped with SeV envelope proteins (SeV-F/HN-SIV) achieved rapid, efficient, and long-lasting gene transfer in the mouse retina. Subretinal exposure to SeV-F/HN-SIV vectors for only a few minutes resulted in high-level gene transfer to the retinal pigment epithelium, whereas several hours were required for gene transfer by standard vesicular stomatitis virus G-pseudotyped SIV vectors. Transgene expression continued over a 1-year period. SeV-F/HN-SIV vector-mediated retinal overexpression of soluble Fms-like tyrosine kinase-1 (sFlt-1) or pigment epithelium-derived factor (PEDF) significantly suppressed laser-induced choroidal neovascularization (CNV). Histologically, 6-month-long sustained overexpression of PEDF did not adversely affect the retina; however, that with sFlt-1 resulted in photoreceptor degeneration associated with choroidal circulation defects. These data demonstrate that brief subretinal administration of SeV-F/HN-SIV vectors may facilitate safe and efficient retinal gene transfer, and suggest the therapeutic potential of PEDF with a higher safety profile for treating CNV in AMD patients. © Copyright 2010, Mary Ann Liebert, Inc.
DOI： 10.1089/hum.2009.102
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Ikeda Y., Yonemitsu Y., Miyazaki M., Kohno R., Murakami Y., Murata T., Goto Y., Tabata T., Ueda Y., Ono F., Suzuki T., Ageyama N., Terao K., Hasegawa M., Sueishi K., Ishibashi T.
Human Gene Therapy 20 ( 9 ) 943 - 954 2009年9月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Human Gene Therapy
A phase 1 clinical trial evaluating the safety of gene therapy for patients with wet age-related macular degeneration (AMD) or retinoblastoma has been completed without problems. The efficacy of gene therapy for Leber's congenital amaurosis (LCA) was reported by three groups. Gene therapy may thus hold promise as a therapeutic method for the treatment of intractable ocular diseases. However, it will first be important to precisely evaluate the efficiency and safety of alternative gene transfer vectors in a preclinical study using large animals. In the present study, we evaluated the acute local (ophthalmic) and systemic toxicity of our simian immunodeficiency virus from African green monkeys (SIVagm)-based lentiviral vectors carrying human pigment epithelium-derived factor (SIV-hPEDF) for transferring genes into nonhuman primate retinas. Transient inflammation and elevation of intraocular pressure were observed in some animals, but these effects were not dose dependent. Electroretinograms (ERGs), including multifocal ERGs, revealed no remarkable change in retinal function. Histopathologically, SIV-hPEDF administration resulted in a certain degree of inflammatory reaction and no apparent structural destruction in retinal tissue. Regarding systemic toxicity, none of the animals died, and none showed any serious side effects during the experimental course. No vector leakage was detected in serum or urine samples. We thus propose that SIVagm-mediated stable gene transfer might be useful and safe for ocular gene transfer in a clinical setting. © Mary Ann Liebert, Inc. 2009.
DOI： 10.1089/hum.2009.048
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Ikeda Y., Yonemitsu Y., Miyazaki M., Kohno R., Murakami Y., Murata T., Tabata T., Ueda Y., Ono F., Suzuki T., Ageyama N., Terao K., Hasegawa M., Sueishi K., Ishibashi T.
Human Gene Therapy 20 ( 6 ) 573 - 579 2009年6月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Human Gene Therapy
Gene therapy may hold promise as a therapeutic approach for the treatment of intractable ocular diseases, including retinitis pigmentosa (RP). Gene transfer vectors that are able to show long-lasting transgene expression in vivo are highly desirable to treat RP; however, there is a dearth of information regarding long-term transgene expression in the eyes of large animals. We previously reported that the simian immunodeficiency virus from African green monkeys (SIVagm)-based lentiviral vector showed efficient, stable, and safe retinal gene transfer, resulting in significant prevention of retinal degeneration by gene transfer of a neurotrophic factor, human pigment epithelium-derived factor (hPEDF), in rodents. Before applying this strategy in a clinical setting, we here assessed the long-lasting transgene expression of our third-generation SIVagm-based lentiviral vectors in the retinal tissue of nonhuman primates. Approximately 20-50μl of SIV-EGFP (enhanced green fluorescent protein) or SIV-hPEDF was injected into the subretinal space via a glass capillary tube. To detect EGFP expression in the retina, we used a fluorescence fundus camera at various time points after gene transfer. Human PEDF expression was assessed by immunohistochemical analysis, Western blot assay, and enzyme-linked immunosorbent assay. The retinas demonstrated frequent EGFP expression that was preserved for at least 4 years without significant decline. The expression of hPEDF was stable, and occurred mainly in the retinal pigment epithelium. The secreted protein was detected in vitreous and aqueous humor. We thus propose that SIVagm-mediated stable gene transfer might be significantly useful for ocular gene transfer in a clinical setting. © Copyright 2009, Mary Ann Liebert, Inc.
DOI： 10.1089/hum.2009.009
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Ex Vivo Transfer of Nuclear Factor-κB Decoy Ameliorates Hepatic Cold Ischemia/Reperfusion Injury 査読あり
Yoshizumi T., Ikeda Y., Kaneda Y., Sueishi K.
Transplantation Proceedings 41 ( 5 ) 1504 - 1507 2009年6月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Transplantation Proceedings
Cold ischemia/reperfusion injury of the hepatic graft has been attributed to the release of various inflammatory cytokines. Specific inhibition of these cytokines may improve viability of the hepatic graft upon reperfusion. Herein we have assessed the efficacy of cis element decoy against nuclear factor-κB binding site delivery to the hepatic tissue in a rodent liver transplantation model. At 8 hours after reperfusion of the liver, significant reduction was noted in the livers treated with decoy in the release of cytosolic enzymes from the hepatocytes and in serum tumor necrosis factor α (P < .05). The neutrophilic infiltration into the hepatic grafts was significantly suppressed in the livers treated with decoy oligodeoxynucleotides (ODNs). Decoy ODNs against nuclear factor-κB binding site delivery improved the viability of the hepatic graft against cold ischemia/reperfusion injury in the rodent liver transplantation model. © 2009 Elsevier Inc. All rights reserved.
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Miyazaki M., Ikeda Y., Yonemitsu Y., Goto Y., Kohno R., Murakami Y., Inoue M., Ueda Y., Hasegawa M., Tobimatsu S., Sueishi K., Ishibashi T.
Journal of Gene Medicine 10 ( 12 ) 1273 - 1281 2008年12月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Gene Medicine
Background: We previously demonstrated that a new lentiviral vector derived from nonpathogenic simian immunodeficiency virus (SIVagm) was efficient and safe for long-lasting retinal gene transfer, and that it provided the significant therapeutic effect of expressing human pigment epithelium-derived factor (hPEDF) in Royal College of Surgeons (RCS) rats. In the present study, to obtain a more pronounced outcome, we assessed the potential synergistic effect of the simultaneous gene transfer of hPEDF and human fibroblast growth factor-2 (hFGF-2) by improved third-generation SIV on RCS rats and retinal degeneration slow (rds) mice, because the former targets the primary neurons, including photoreceptor cells (PCs), whereas the latter is effective for targeting secondary neural cells, including Muller cells. Methods: Vector solution (SIV-hPEDF, SIV-hFGF-2, a 1:1 mixture of SIV-hPEDF and SIV-hFGF-2, or SIV-enhanced green fluorescent protein) was injected into the peripheral subretinal space of 3-week-old RCS rats or rds mice. Histopathological and electroretinographic assessments were made at several points after gene transfer. Results: Administration of SIV-hPEDF or SIV-hFGF-2 significantly delayed the histological PC degeneration and electrical deficit in RCS rats, and these delays were synergistically and significantly pronounced by SIV-hPEDF + SIV-hFGF-2 (1:1 mixture). In rds mice, functional therapeutic effects were observed even by SIV-PEDF, or SIV-FGF-2 alone and, moreover, both SIV-PEDF and SIV-FGF-2 showed higher therapeutic effects. Conclusions: These synergistic rescues of retinitis pigmentosa (RP) model animals are the 'proof concept' that the 'dual' expression of hPEDF and hFGF-2 dramatically improved therapeutic efficacy by keeping lower titers. This strategy may contribute to safer and more effective gene therapy for RP. Copyright © 2008 John Wiley & Sons, Ltd.
DOI： 10.1002/jgm.1257
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Murakami Y., Ikeda Y., Yonemitsu Y., Onimaru M., Nakagawa K., Kohno R., Miyazaki M., Hisatomi T., Nakamura M., Yabe T., Hasegawa M., Ishibashi T., Sueishi K.
American Journal of Pathology 173 ( 5 ) 1326 - 1338 2008年11月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：American Journal of Pathology
Photoreceptor apoptosis is a critical process of retinal degeneration in retinitis pigmentosa (RP), a group of retinal degenerative diseases that result from rod and cone photoreceptor cell death and represent a major cause of adult blindness. We previously demonstrated the efficient prevention of photoreceptor apoptosis by intraocular gene transfer of pigment epithelium-derived factor (PEDF) in animal models of RP; however, the underlying mechanism of the neuroprotective activity of PEDF remains elusive. In this study, we show that an apoptosis-inducing factor (AIF)-related pathway is an essential target of PEDF-mediated neuroprotection. PEDF rescued serum starvation-induced apoptosis, which is mediated by AIF but not by caspases, of R28 cells derived from the rat retina by preventing translocation of AIF into the nucleus. Nuclear translocation of AIF was also observed in the apoptotic photoreceptors of Royal College of Surgeons rats, a well-known animal model of RP that carries a mutation of the Mertk gene. Lentivirus-mediated retinal gene transfer of PEDF prevented the nuclear translocation of AIF in vivo, resulting in the inhibition of the apoptotic loss of their photoreceptors in association with up-regulated Bcl-2 expression, which mediates the mitochondrial release of AIF. These findings clearly demonstrate that AIF is an essential executioner of photoreceptor apoptosis in inherited retinal degeneration and provide a therapeutic rationale for PEDF-mediated neuroprotective gene therapy for individuals with RP. Copyright © American Society for Investigative Pathology.
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Murakami Y., Ikeda Y., Yonemitsu Y., Tanaka S., Kondo H., Okano S., Kohno R., Miyazaki M., Inoue M., Hasegawa M., Ishibashi T., Sueishi K.
Journal of Gene Medicine 10 ( 2 ) 165 - 176 2008年2月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Gene Medicine
Background: Recombinant Sendai virus vectors (rSeV) constitute a new class of cytoplasmic RNA vectors that have shown efficient gene transfer in various organs, including retinal tissue; however, the related immune responses remain to be overcome in view of clinical applications. We recently developed a novel rSeV from which all envelope-related genes were deleted (rSeV/dFdMdHN) and, in the present study, assess host immune responses following retinal gene transfer. Methods: rSeV/dFdMdHN or conventional F-gene deleted rSeV (rSeV/dF) was injected into subretinal space of adult Wistar rats or C57BL/6 mice. The transgene expression and histopathological findings were assessed at various time points. Immunological assessments, including the expression of proinflammatory cytokines, natural killer (NK)-cell activity, as well as SeV-specific cytotoxic T lymphocytes (CTLs) and antibodies, were performed following vector injection. Results: rSeV/dFdMdHN showed high gene transfer efficiency into the retinal pigment epithelium at an equivalent level to that seen with rSeV/ dF. In the early phase, the upregulation of proinflammatory cytokines, local inflammatory cell infiltration and tissue damage that were all prominently seen in rSeV/dF injection were dramatically diminished using rSeV/dFdMdHN. NK cell activity was also decreased, indicating a reduction of the innate immune response. In the later phase, on the other hand, CTL activity and anti-SeV antibodies were similarly induced, even using rSeV/dFdMdHN, and resulted in transient transgene expression in both vector types. Conclusions: Deletion of envelope-related genes of rSeV dramatically reduces the vector-induced retinal damage and may extend the utility for ocular gene transfer; however, further studies regulating the acquired immune response are required to achieve long-term transgene expression of rSeV. Copyright © 2007 John Wiley & Sons, Ltd.
DOI： 10.1002/jgm.1142
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Interleukin-18 regulates pathological intraocular neovascularization 査読あり
Qiao H., Sonoda K., Ikeda Y., Yoshimura T., Hijioka K., Jo Y., Sassa Y., Tsutsumi-Miyahara C., Hata Y., Akira S., Ishibashi T.
Journal of Leukocyte Biology 81 ( 4 ) 1012 - 1021 2007年4月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Leukocyte Biology
Recently, the proinflammatory cytokine IL-18 has been shown to have a role in angiogenesis. This study aimed to elucidate its role in abnormal neovascularization (NV) in an oxygen-induced retinopathy (OIR) mouse model of the retinopathy seen in human premature newborns. IL-18 was constitutively expressed in the retina in C57BL/6 mice, but expression transiently dropped on Day 17 after birth in mice exposed to 75% oxygen for 5 days between Days 7 and 12. Coincident with the IL-18 reduction in oxygen-treated mice, vascular endothelial growth factor was expressed in the retina, and OIR developed. By Day 24, NV in the retina had regressed to normal levels. By contrast, IL-18 knockout mice, exposed to elevated oxygen concentrations, developed more severe OIR on Day 17, and it is important that this persisted until Day 24. This suggested that IL-18 negatively regulated retinal NV. To investigate this further, we administrated recombinant IL-18 to C57BL/6 mice during the development of OIR but found no significant inhibition of retinopathy. However, when IL-18-binding protein was administered during the OIR recovery phase to neutralize endogenous IL-18, OIR was still apparent on Day 24. We therefore concluded that IL-18 regulates pathogenic retinal NV by promoting its regression rather than inhibiting its development. This suggests some useful, new approaches to treating retinopathy in humans. © Society for Leukocyte Biology.
DOI： 10.1189/jlb.0506342
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Rapid detection of SAG 926delA mutation using real-time polymerase chain reaction 査読あり
Yoshida S., Yamaji Y., Yoshida A., Ikeda Y., Yamamoto K., Ishibashi T.
Molecular Vision 12 1552 - 1557 2006年12月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Molecular Vision
Purpose: Mutation 926delA of the arrestin/S-antigen SAG gene is the main cause of Oguchi disease in the Japanese. The purpose of this study was to develop a rapid diagnostic assay to detect mutations in the SAG gene. Methods: Two sequence-specific primers and fluorophore-labeled probes for exon 11 of the SAG gene were designed, and the region spanning the mutations was amplified by polymerase chain reaction (PCR) using the LightCycler detection system (Roche Diagnostics, Mannheim, Germany). The mutations were then identified by melting curve analyses of the hybrid formed between the PCR product and a specific fluorescent probe. Results: We clearly distinguished each SAG genotype (homozygous and heterozygous 926delA and wild type) by the distinct melting peaks at different temperatures. One thermal cycling required approximately 54 min to process, and the results were 100% in concordance with the genotypes determined by DNA sequencing. Conclusions: We have succeeded in developing a rapid method to detect the most frequent mutation in the SAG gene. This method will help in identifying gene mutations associated with Oguchi disease with a rapid and reliable identification or the exclusion of the frequent mutations in the SAG gene. © 2006 Molecular Vision.
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Ikeda Y., Yonemitsu Y., Onimaru M., Nakano T., Miyazaki M., Kohno R., Nakagawa K., Ueno A., Sueishi K., Ishibashi T.
Experimental Eye Research 83 ( 5 ) 1031 - 1040 2006年11月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Experimental Eye Research
The vascular endothelial growth factor (VEGF) family plays an essential role in vascular development, angiogenesis and lymphangiogenesis. VEGF-A is a key regulator of endothelial cell functions and VEGF-C and VEGF-D are known to stimulate both angiogenesis and lymphangiogenesis. In a surgically removed subretinal vascular membrane of an age-related macular degeneration (AMD) patient, both VEGF-C and VEGF-D were confirmed, in addition to VEGF-A, to be markedly positive in the retinal pigment epithelium (RPE). There is no lymph vessel in ocular tissue, so it is possible that VEGF-C and VEGF-D expression in the RPE play some role in ocular angiogenesis, as well as VEGF-A. Next, we assessed the transition of VEGF-A, -C, and -D expression on several conditions, in human RPE. Hypoxia proverbially induced VEGF-A mRNA expression, meanwhile VEGF-C and VEGF-D mRNA expression was down-regulated. The Ca2+ deprivation from culture medium strongly up-regulated VEGF-A and VEGF-D mRNA expression. Culture on plastic flasks precoated with poly-2-hydroxyethyl methacrylate up-regulated VEGF-D expression. Meanwhile, no significant change of VEGF-C mRNA expression was found in the blockade of cell-cell and/or cell-matrix adhesion. These findings suggest the possibility that VEGF-C and VEGF-D expression in RPE modify the ocular angiogenesis as angiogenic stimulators. © 2006 Elsevier Ltd. All rights reserved.
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Kohno R., Ikeda Y., Yonemitsu Y., Hisatomi T., Yamaguchi M., Miyazaki M., Takeshita H., Ishibashi T., Sueishi K.
Brain Research 1093 ( 1 ) 54 - 70 2006年6月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Brain Research
It is well known that neural stem/progenitor cells of the central nervous system (CNS) can proliferate to form neurospheres (CNS-neurospheres) that are positive for nestin, an intermediate filament for neural progenitors. Retinal stem/progenitor properties were also isolated from the ciliary body (CB) of the eye where, as in the CNS, such stem/progenitors also form spheres and have been considered to expand only via expansion by their proliferation even from the single-cell level (called spheres of pigment cells from the ciliary margin: PCM-spheres). We here found a new and distinct process underlying the growth of CB cell-derived spheres (CB-spheres) that is unlike the mechanism of CNS- and PCM-sphere expansion; this new process is a cell proliferation-independent incorporation of neighbor spheres and cells cultured at high density (200 cells/μl). The majority of cells in CB-spheres consisted of nestin-negative epithelia-like cells and started to express nestin during the course of their expansion by high-density cultivation. The growth of CNS-neurospheres was sensitive to a cell-cycle inhibitor, whereas the growth of CB-spheres was not seriously affected by cell proliferation; rather, the spheres grew by incorporating other CB-spheres and nestin-negative adherent cells, the latter of which started to express nestin and lost the expression of epithelial markers after being incorporated. These results indicate that CB-spheres do not form by the accumulation of neural progenitors but rather by a reprogramming system from epithelia-like cells for neural differentiation, a clearly distinct mechanism from sphere formation by single-cell expansion of retinal stem/progenitor populations. © 2006 Elsevier B.V. All rights reserved.
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Tumor necrosis factor-α antisense transfer remarkably improves hepatic graft viability 査読あり
Yoshizumi T., Yonemitsu Y., Ikeda Y., Kaneda Y., Yanaga K., Sugimachi K., Sueishi K.
Liver International 26 ( 4 ) 451 - 456 2006年5月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Liver International
Background: Cold ischemia/reperfusion injury of the hepatic graft, an unsolved problem in liver transplantations, is attributed to the release of inflammatory cytokines, especially the tumor necrosis factor- (TNF) α, from activated Kupffer cells (KC). Therefore, the specific inhibition of TNF-α could improve the viability of the hepatic graft upon reperfusion.: We assessed the efficacy of TNF-α antisense (TNF-AS) oligodeoxynucleotides (ODNs) delivery to KC in a rodent liver transplantation model. Results: Seventy-one percent of the animals that received 6 hours preserved grafts in baths of lactated Ringer's solution (4°C) and were treated with TNF-AS survived for over 14 days. Eighty percent of the animals treated with vehicle, sense ODNs, or balanced salt saline (BSS) died. Four hours after reperfusion of the liver, a significant reduction was noted in livers treated with TNF-AS in the release of cytosolic enzymes from the hepatocytes and the serum TNF-α (P<0.05). The expressions of TNF-α on KC and of intercellular adhesion molecule-1 on sinusoidal endothelial cells were completely suppressed in TNF-AS-treated livers. Conclusions: TNF-AS delivery improves the viability of the hepatic graft, and this technique may solve hepatic graft nonfunction in a clinical setting. © 2006 Blackwell Munksgaard.
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Baba H., Yonemitsu Y., Nakano T., Onimaru M., Miyazaki M., Ikeda Y., Sumiyoshi S., Ueda Y., Hasegawa M., Yoshino I., Maehara Y., Sueishi K.
Arteriosclerosis, Thrombosis, and Vascular Biology 25 ( 9 ) 1938 - 1944 2005年9月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Arteriosclerosis, Thrombosis, and Vascular Biology
Objective - To assess the expression and distribution of a neurotrophic/antiangiogenic factor, pigment epithelium-derived factor (PEDF), related to angiogenesis that is a possibly key event during atherogenesis in human atherosclerotic plaques. Methods and Results - Twenty fresh aortic samples were used for reverse-transcription polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry (IHC). In addition, 80 stocked paraffin blocks of coronary arteries from 40 autopsy cases were also used. IHC revealed divergent staining patterns for PEDF in both the aortas and the coronary arteries tested, ie, "cytoplasmic staining" or "extracellular deposition," were observed, respectively. In the areas showing cytoplasmic staining, double PEDF was expressed in a majority of the foamy macrophages and in some smooth muscle cells, and the PEDF-positive cell frequency was positively correlated with that of microvessels in a cell-rich area in the coronary arteries (P<0.0001). Inversely, extracellular deposition of PEDF was seen in acellular areas and was negatively correlated with the number of microvessels (P=0.0003). Conclusions - These results suggest that PEDF may function as an antiangiogenic factor when it is deposited onto the extracellular matrix. Thus, PEDF may play a significant role in determining the balance of angiogenesis/antiangiogenesis during atherogenesis. © 2005 American Heart Association, Inc.
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Abnormal retinal vascular development in IL-18 knockout mice 査読あり
Qiao H., Sonoda K., Sassa Y., Hisatomi T., Yoshikawa H., Ikeda Y., Murata T., Akira S., Ishibashi T.
Laboratory Investigation 84 ( 8 ) 973 - 980 2004年8月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Laboratory Investigation
Recent studies have indicated that interleukin 18 (IL-18) might act as either an angiogenic or an angiostatic factor, but the true function of this protein in vascular development is unclear. We therefore investigated the role of IL-18 in the formation of retinal vessels. Development of the retinal vasculature was compared in IL-18 knockout (KO) and wild-type (WT) mice at several different time points. The formation of vessels was evaluated using angiography of flat-mounted retinal samples after inoculation with fluorescein dextran. Retinal samples from both groups were also evaluated through histological examinations, and the expression of angiogenic factors was examined using the reverse-transcription-polymerase chain reaction. The capillary retinal vessels in both WT and IL-18 KO mice had reached the peripheral retina by postnatal day (P) 7. However, IL-18 KO mice showed angiectasis and vascular leakage at P7, especially in the mid-peripheral retina. These symptoms were not observed in WT mice at any stage. Histopathological analysis confirmed abnormal vascular formation in IL-18 KO mice at P14. Interestingly, these abnormalities regressed over time and had disappeared by P84. Several angiogenesis-associated factors, including vascular enclothelial growth factor (VEGF), basic fibroblast-growth factor (bFGF), platelet-derived growth factor (PDGF) and pigment epithelium-derived factor (PEDF), were overexpressed in the retinas of IL-18 KO mice compared with those of WT mice at P14. Interferon-γ was detected only in WT mouse retinas at P14. These results provide new evidence for the role of IL-18 in retinal vascular development.
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Miyazaki M., Ikeda Y., Yonemitsu Y., Goto Y., Sakamoto T., Tabata T., Ueda Y., Hasegawa M., Tobimatsu S., Ishibashi T., Sueishi K.
Gene Therapy 10 ( 17 ) 1503 - 1511 2003年8月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Gene Therapy
Retinitis pigmentosa (RP) is a heterogenous group of inherited retinal diseases resulting in adult blindness caused by mutations of various genes. Although it is difficult to cure the blindness that results from these diseases, delaying the disease progression may be of great benefit, since the majority of RP diseases are seen in middle age or later. To test a gene therapy strategy for RP using a neurotrophic factor gene, we assessed the effect of simian lentivirus (SIV)-mediated subretinal gene transfer of pigment epithelium-derived factor (PEDF), a potent neurotrophic factor, during the disease progression in Royal College of Surgeons (RCS) rats, a well-accepted animal model of RP. Regional gene transfer via SIV into the peripheral subretinal space at the nasal hemisphere was performed in all animals to monitor site-specific transgene expression as well as the therapeutic effect in each retina. Gene transfer of lacZ and PEDF was observed in the regional pigment epithelium corresponding to the regional gene transfer. Histologically, PEDF gene transfer significantly protected the loss of photoreceptor cells (PCs) corresponding to the regions of the gene transfer, compared to those of control groups during the course of the experiment. The antiapoptotic effect of PEDF on PCs is likely to be a related mechanism, because a significant reduction of terminal dUTP-nicked end labeling-positive PC numbers was found in PEDF-treated eyes compared to those of the control group (P < 0.05). PEDF-treated eyes also retained a significant sensitivity to light flash during the experimental course. These findings clearly show that neuroprotective gene therapy using PEDF can protect retinal degeneration and functional defects in individuals with RP.
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Ikeda Y., Goto Y., Yonemitsu Y., Miyazaki M., Sakamoto T., Ishibashi T., Tabata T., Ueda Y., Hasegawa M., Tobimatsu S., Sueishi K.
Gene Therapy 10 ( 14 ) 1161 - 1169 2003年7月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Gene Therapy
Although lentivirus vectors hold promise for ocular gene therapy, they also have potential safety issues, particularly in the case of the current human immunodeficiency virus-based vectors. We recently developed a novel lentivirus vector derived from the nonpathogenic simian immunodeficiency virus from African green monkeys (SIVagm) to minimize these potentials. In this preclinical study, we evaluated whether SIV vector could be efficiently and safely applicable to retinal gene transfer by assessing the transgene expression, retinal function and histology over a 1-year period following subretinal injection in adult rats. The functional assessment via electroretinogram after both titers of SIV-lacZ (2.5 × 107 or 2.5 × 108 transducing units/ml) injection revealed both the dark and light adaptations to soon be impaired, in a dose-dependent manner, after a buffer injection as well, and all of them recovered to the control range by day 30. In both titers tested, the retinas demonstrated a frequent transgene expression mainly in the retinal pigment epithelium; however, the other retinal cells rarely expressed the transgene. Retinas exposed to a low titer virus showed no significant inflammatory reaction throughout the observation period, and also maintained the transgene expression over a 1-year period. In the retinas exposed to a high titer virus, however, mononuclear cell infiltration persisted in the subretinal area, and the retina that corresponded to the injected area finally underwent degeneration by around day 90. No retinal neoplastic lesions could be found in any animals over the 1-year period. We thus propose that SIV-mediated stable gene transfer might be useful for ocular gene transfer, however, more attention should be paid to avoiding complications when administering high titer lentivirus to the retina.
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Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary hypertension in rats 査読あり
Ikeda Y., Yonemitsu Y., Kataoka C., Kitamoto S., Yamaoka T., Nishida K., Takeshita A., Egashira K., Sueishi K.
American Journal of Physiology - Heart and Circulatory Physiology 283 ( 5 52-5 ) 2002年11月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：American Journal of Physiology - Heart and Circulatory Physiology
Monocyte/macrophage chemoattractant protein-1 (MCP-1), a potent chemoattractant chemokine and an activator for mononuclear cells, may play a role in the initiation and/or progression of pulmonary hypertension (PH). To determine whether blockade of a systemic MCP-1 signal pathway in vivo may prevent PH, we intramuscularly transduced a naked plasmid encoding a 7-NH 2 terminus-deleted dominant negative inhibitor of the MCP-1 (7ND MCP-1) gene in monocrotaline-induced PH. We also simultaneously gave a duplicate transfection at 2-wk intervals or skeletal muscle-directed in vivo electroporation (EP) to evaluate whether a longer or higher expression might be more effective. The intramuscular reporter gene expression was enhanced 10 times over that by EP than by simple injection, and a significant 7ND MCP-1 protein in plasma was detected only in the EP group. 7ND MCP-1 gene transfer significantly inhibited the progression of MCT-induced PH as evaluated by right ventricular systolic pressure, right ventricular hypertrophy, medial hypertrophy of pulumonary arterioles, and mononuclear cell infiltration into the lung. Differential effects of longer or higher transgene expression were not apparent. Although the in vivo kinetics of 7ND MCP-1 gene therapy should be studied further, these encouraging results suggest that an anti-inflammatory strategy via blockade of the MCP-1 signal pathway may be an alternative approach to treat subjects with PH.
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Ikeda Y., Yonemitsu Y., Sakamoto T., Ishibashi T., Ueno H., Kato A., Nagai Y., Fukumura M., Inomata H., Hasegawa M., Sueishi K.
Experimental Eye Research 75 ( 1 ) 39 - 48 2002年
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Experimental Eye Research
To determine the usefulness of recombinant Sendai virus (SeV) for ocular gene transfer, the authors characterized SeV-mediated gene transfer to the retinal tissue of adult rats via subretinal injection. Recombinant SeV encoding the lacZ gene achieved frequent transgene expression in the retinal pigment epithelium (RPE) (mean = 38.76%), while gene transfer to other retinal cells was rare. These findings are similar to those of previous reports using adenoviruses. Peak reporter gene expression of SeV in cultured RPE cells was similar to that of adenovirus at the same titer; however, SeV achieved high levels of expression after a brief vector-cell contact time, while adenovirus required over 3 hr for efficient gene transfer. This finding was also observed in vivo following a brief SeV filling in the subretinal space, and may therefore provide a clinical advantage in avoiding retinal damage due to prolonged detachment. The observed SeV-mediated gene expression in the rat retina was transient. The initial phase of the decrease in luciferase activity could be prevented by daily eye drops of dexamethasone, suggesting that the corticosteroid-sensitive host reaction may affect early clearance of the virus. The late decline of transgene expression (2 weeks) was inhibited by the immunosuppressant, cyclosporin A, in a dose-dependent manner, suggesting that the cytotoxic T-lymphocyte response may be important in this phase. This work represents the first report of SeV-mediated gene transfer to ocular tissue, and identifies recombinant SeV as a new tool for studies of retinal gene transfer and gene therapy. © 2002 Elsevier Science Ltd.
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Gene targeting to the retina 査読あり
Sakamoto T., Ikeda Y., Yonemitsu Y.
Advanced Drug Delivery Reviews 52 ( 1 ) 93 - 102 2001年10月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Advanced Drug Delivery Reviews