[Show abstract][Hide abstract] ABSTRACT: After the first report of induced pluripotent stem cells (iPSCs), considerable efforts have been made to develop more efficient methods for generating iPSCs without foreign gene insertions. Here we show that Sendai virus vector, an RNA virus vector that carries no risk of integrating into the host genome, is a practical solution for the efficient generation of safer iPSCs. We improved the Sendai virus vectors by introducing temperature-sensitive mutations so that the vectors could be easily removed at nonpermissive temperatures. Using these vectors enabled the efficient production of viral/factor-free iPSCs from both human fibroblasts and CD34(+) cord blood cells. Temperature-shift treatment was more effective in eliminating remaining viral vector-related genes. The resulting iPSCs expressed human embryonic stem cell markers and exhibited pluripotency. We suggest that generation of transgene-free iPSCs from cord blood cells should be an important step in providing allogeneic iPSC-derived therapy in the future.
Proceedings of the National Academy of Sciences 08/2011; 108(34):14234-9. DOI:10.1073/pnas.1103509108 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lentiviral vectors are promising tools for the treatment of chronic retinal diseases including glaucoma, as they enable stable transgene expression. We examined whether simian immunodeficiency virus (SIV)-based lentiviral vector-mediated retinal gene transfer of human pigment epithelium-derived factor (hPEDF) can rescue rat retinal ganglion cell injury. Gene transfer was achieved through subretinal injection of an SIV vector expressing human PEDF (SIV-hPEDF) into the eyes of 4-week-old Wistar rats. Two weeks after gene transfer, retinal ganglion cells were damaged by transient ocular hypertension stress (110 mmHg, 60 min) and N-methyl-d-aspartic acid (NMDA) intravitreal injection. One week after damage, retrograde labeling with 4',6-diamidino-2-phenylindole (DAPI) was done to count the retinal ganglion cells that survived, and eyes were enucleated and processed for morphometric analysis. Electroretinographic (ERG) assessment was also done. The density of DAPI-positive retinal ganglion cells in retinal flat-mounts was significantly higher in SIV-hPEDF-treated rats compared with control groups, in both transient ocular hypertension and NMDA-induced models. Pattern ERG examination demonstrated higher amplitude in SIV-hPEDF-treated rats, indicating the functional rescue of retinal ganglion cells. These findings show that neuroprotective gene therapy using hPEDF can protect against retinal ganglion cell death, and support the potential feasibility of neuroprotective therapy for intractable glaucoma.
Human gene therapy 12/2010; 22(5):559-65. DOI:10.1089/hum.2010.132 · 3.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gene therapy for cystic fibrosis (CF) is making encouraging progress into clinical trials. However, further improvements in transduction efficiency are desired. To develop a novel gene transfer vector that is improved and truly effective for CF gene therapy, a simian immunodeficiency virus (SIV) was pseudotyped with envelope proteins from Sendai virus (SeV), which is known to efficiently transduce unconditioned airway epithelial cells from the apical side. This novel vector was evaluated in mice in vivo and in vitro directed toward CF gene therapy. Here, we show that (i) we can produce relevant titers of an SIV vector pseudotyped with SeV envelope proteins for in vivo use, (ii) this vector can transduce the respiratory epithelium of the murine nose in vivo at levels that may be relevant for clinical benefit in CF, (iii) this can be achieved in a single formulation, and without the need for preconditioning, (iv) expression can last for 15 months, (v) readministration is feasible, (vi) the vector can transduce human air-liquid interface (ALI) cultures, and (vii) functional CF transmembrane conductance regulator (CFTR) chloride channels can be generated in vitro. Our data suggest that this lentiviral vector may provide a step change in airway transduction efficiency relevant to a clinical programme of gene therapy for CF.
[Show abstract][Hide abstract] ABSTRACT: 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.
Human gene therapy 06/2009; 20(9):943-54. DOI:10.1089/hum.2009.048 · 3.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract 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 20-50 mul 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.
Human gene therapy 04/2009; 20(6):573-9. DOI:10.1089/hum.2009.009 · 3.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Platelets release several mediators that modify vascular integrity and hemostasis. In the present study, we developed a technique for efficient transgene expression in platelets in vivo and examined whether this targeted-gene-product delivery system using a platelet release reaction could be exploited for clinical applications. Analysis of luciferase reporter gene constructs driven by platelet-specific promoters (the GPIIb, GPIbalpha, and GPVI) revealed that the GPIbalpha promoter was the most potent in the megakaryoblastic cell line UT-7/TPO and human CD34+-derived megakaryocytes. Transduction of UT-7/TPO; CD34+-derived megakaryocytes; and c-Kit+, ScaI+, and Lineage- (KSL) murine hematopoietic stem cells with a simian immunodeficiency virus (SIV)-based lentiviral vector carrying eGFP resulted in efficient, dose-dependent expression of eGFP, and the GPIbalpha promoter seemed to bestow megakaryocytic-specific expression. Transplantation of KSL cells transduced with SIV vector containing eGFP into mice showed that there was preferable expression of eGFP in platelets driven by the GPIbalpha promoter [7-11% for the cytomeglovirus (CMV) promoter, 16-27% for the GPIbalpha promoter]. Furthermore, transplantation of ex vivo-transduced KSL cells by SIV vector carrying human factorVIII (hFVIII) driven by the GPIbalpha promoter induced the production of detectable transcripts of the hFVIII gene and the hFVIII antigen in bone marrow and spleen for at least 90 days and partially corrected the hemophilia A phenotype. Platelet-targeting gene therapy using SIV vectors appears to be promising for gene therapy approaches toward not only inherited platelet diseases but also other hemorrhagic disorders such as hemophilia A.
The FASEB Journal 08/2006; 20(9):1522-4. DOI:10.1096/fj.05-5161fje · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously shown that recombinant Sendai-virus (SeV) vector transduces airway epithelium efficiently via the apical membrane. This is in part due to cholesterol and sialic acid in the apical membrane which are respectively utilized by the SeV fusion (F) and hemagluttinin-neuraminidase (HN) proteins for receptor- mediated cell attachment, as well as the capacity of SeV to penetrate mucus efficiently. In contrast to SeV, which leads to transient gene expression, integrating lentiviral vectors have the theoretical advantage of persistent expression for the life-time of the cell, and ifstem cell integration should occur, for the lifetime of the animal. We have recently succeeded in generating a F/HN-pseudotyped Simian immunodeficiency virus (SiVagm) vector at high enough titre for in vivo application (>109 transduction units (TU)/ml). A F/HN-SiV- GFP vector (4x108 TU/mouse in 100 ml) was slowly perfused (over 75 min) onto the nasal epithelium of C57Bl/6 mice and transduced airway epithelial cells efficiently, via the apical membrane without the need for chemical pre-conditioning. We choose transduction of the nasal epithelium as proof-of-principle because we aim to extend these studies into CF knockout mice (only the nasal epithelium shows the CF defect in this model). We previously reported detection of GFP expression at 1, 20, 30, 90 days (n=3 mice/group) and 220 days (n=1) after transduction. Here, we further extended these studies to both include higher n numbers and, to identify transduced cells. Importantly, 180 days after transduction 8 out of 10 (6 histological sections 1 mm apart/mouse) mice still expressed GFP. Preliminary experiments on a single animal also showed persistent expression 360 days after transduction, and further animals transduced 270 and 360 days ago, are currently being analysed (n=12–15/group). This duration of gene expression is far longer than the predicted life-span of airway epithelial cells of approximately 100 days. One explanation is that F/HN-SiV vector has transduced progenitor or stem cells in the mouse nose. Morphological analysis showed that GFP positive cells were ciliated airway epithelial cells, and cells being in the olfactory epithelium which based on morphological criteria, appeared to be sensory neuronal cells.
[Show abstract][Hide abstract] ABSTRACT: Purpose: Recently, we have demonstrated the efficient and stable retinal gene transfer mediated by the non-pathogenic simian immunodeficiency virus (SIV)-based lentivirus vector as well as the therapeutic outcome in some animal models of retinal degeneration using recombinant SIV vectors carrying neurotrophic factors, such as pigment epithelium-derived factor (PEDF) and fibroblast growth factor-2 (FGF-2). Here, we report the acute ophthalmic toxicity following intraocular administration of SIV-PEDF in Macaca fascicularis, as a preclinical safety study.Methods: Eleven Macaca fascicularis (body weight=2.35-5.63 kg) were enrolled in this study. Approximately 20 μl of SIV-PEDF (low titer: 2.5×107 transducing units [TU]/ml, equal to clinically available titer, high titer: 2.5×108 TU)/ml, and max titer: 1.0×109 TU/ml, n=3, respectively) or control (balanced salt solution: BSS, n=2) were injected into subretinal space via a glass capillary tube. We undertook an ophthalmic examination including slitlamp biomicroscopy, intraocular pressure (IOP) measurement, fundoscopic examination, and fluorescein anigiography, and assessed transgene expression, retinal function with electroretinogram (ERG) and histology for 3 months.Results: SIV vector was efficiently transferred mainly to the retinal pigment epithelium and transgene expression was detected within 3 months. Transient inflammatory cell infiltration to anterior ocular segment and elevation of IOP were observed in some animals, but not dose-dependent. ERGs including multi-focal ERGs revealed no remarkable change of the retinal functions. Retinas treated with both SIV-PEDF and BSS showed no significant inflammatory infiltration and retinal structural destruction. Furthermore, neither dead animal nor serious side effect was found during experimental course.Conclusions: The current study indicated the acute local and systemic safety of intraocular administration of SIV-PEDF, even if it was given at 40-times higher titer than clinical available titer. We are now conducting the long-termed safety study in non-human primates.
[Show abstract][Hide abstract] ABSTRACT: The successful engraftment of genetically modified hematopoietic stem cells (HSCs) without toxic conditioning is a desired goal for HSC gene therapy. To this end, we have examined the combination of intrabone marrow transplantation (iBMT) and in vivo expansion by a selective amplifier gene (SAG) in a nonhuman primate model. The SAG is a chimeric gene consisting of the erythropoietin (EPO) receptor gene (as a molecular switch) and c-Mpl gene (as a signal generator). Cynomolgus CD34+ cells were retrovirally transduced with or without SAG and returned into the femur and humerus following irrigation with saline without prior conditioning. After iBMT without SAG, 2-30% of colony-forming cells were gene marked over 1 year. The marking levels in the peripheral blood, however, remained low (<0.1%). These results indicate that transplanted cells can engraft without conditioning after iBMT, but in vivo expansion is limited. On the other hand, after iBMT with SAG, the peripheral marking levels increased more than 20-fold (up to 8-9%) in response to EPO even at 1 year posttransplant. The increase was EPO-dependent, multilineage, polyclonal, and repeatable. Our results suggest that the combination of iBMT and SAG allows efficient in vivo gene transduction without marrow conditioning.
[Show abstract][Hide abstract] ABSTRACT: Gene therapy is being studied as the next generation therapy for hemophilia and several clinical trials have been carried out, albeit with limited success. To explore the possibility of utilizing autologous bone marrow transplantation of genetically modified hematopoietic stem cells for hemophilia gene therapy, we investigated the efficacy of genetically engineered CD34+ cell transplantation to NOD/SCID mice for expression of human factor VIII (hFVIII).
CD34+ cells were transduced with a simian immunodeficiency virus agmTYO1 (SIV)-based lentiviral vector carrying the enhanced green fluorescent protein (eGFP) gene (SIVeGFP) or the hFVIII gene (SIVhFVIII). CD34+ cells transduced with SIV vectors were transplanted to NOD/SCID mice. Engraftment of transduced CD34+ cells and expression of transgenes were studied.
We could efficiently transduce CD34+ cells using the SIVeGFP vector in a dose-dependent manner, reaching a maximum (99.6 +/- 0.1%) at MOI of 5 x 10(3) vector genome/cell. After transducing CD34+ cells with SIVhFVIII, hFVIII was produced (274.3 +/- 20.1 ng) from 10(6) CD34+ cells during 24 h in vitro incubation. Transplantation of SIVhFVIII-transduced CD34+ cells (5-10 x 10(5)) at a multiplicity of infection (MOI) of 50 vector genome/cell into NOD/SCID mice resulted in successful engraftment of CD34+ cells and production of hFVIII (minimum 1.2 +/- 0.9 ng/mL, maximum 3.6 +/- 0.8 ng/mL) for at least 60 days in vivo. Transcripts of the hFVIII gene and the hFVIII antigen were also detected in the murine bone marrow cells.
Transplantation of ex vivo transduced hematopoietic stem cells by non-pathogenic SIVhFVIII without exposure of subjects to viral vectors is safe and potentially applicable for gene therapy of hemophilia A patients.
The Journal of Gene Medicine 10/2004; 6(10):1049-60. DOI:10.1002/jgm.609 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It has recently been reported that bone marrow cells can efficiently engraft without marrow conditioning when implanted directly into the bone marrow cavity (intra-bone marrow transplantation, iBMT) in mice. We have successfully examined the efficacy of autologous iBMT in a cynomolgus monkey model in conjuction with an in vivo expansion of transplanted cells by a selective amplifier transgene (Ueda et al., 2004) and provide here the detailed parameters of our iBMT method. We injected retrovirally-marked autologous CD34+ cells directly into the non-conditioned marrow cavity of the femur and humerus after gently irrigating the cavity with saline. This transplant procedure was safely performed without pulmonary embolism. Gene-marked cells were not detectable in the peripheral blood at one hour and one day after iBMT as assessed by sensitive PCR, indicating that iBMT hardly generated a systemic delivery of transplanted cells. On the other hand, 2 to 30% of clonogenic hematopoietic colonies produced from the implanted marrow were gene-marked at 612 months after iBMT. Our iBMT method for non-human primates is thus discussed in terms of long-lived hematopoietic stem/progenitor cells, bone marrow niche and long-term engraftment after iBMT without myeloablative conditioning.
Gene Therapy and Regulation 07/2004; 2(3):207-218. DOI:10.1163/1568558042457460
[Show abstract][Hide abstract] ABSTRACT: We demonstrate that transduction of adipocytes with a simian immunodeficiency virus agm TYO1 (SIVagm)-based lentiviral vector carrying the human coagulation factor VIII gene (SIVhFVIII) resulted in expression of the human FVIII transgene in vitro and in db/db mice in vivo. Cultured human adipocytes were transduced with the SIVagm vector carrying the GFP gene in a dose-dependent manner and transduction of adipocytes with SIVhFVIII resulted in efficient expression of human coagulation factor VIII (hFVIII; 320 +/- 39.8 ng/10(6) adipocytes/24 h) in vitro. Based upon successful transduction of adipocytes by SIV vectors carrying the lacZ gene in vivo in mice, the adipose tissue of db/db mice was transduced with SIVhFVIII. There was a transient appearance of human FVIII in mouse plasma (maximum 1.8 ng/ml) on day 11 after the injection. Transcripts of human FVIII transgene and human FVIII antigen also were detected in the adipose tissue by RT-PCR and immunofluorescence, respectively, on day 14. Emergence of anti-human FVIII antibodies 14 days after the injection of SIVhFVIII may explain the disappearance of human FVIII from the circulation. These results suggest that transduction of the adipocytes with vectors carrying the human FVIII gene may be potentially applicable for gene therapy of hemophilia A.
[Show abstract][Hide abstract] ABSTRACT: The successful hematopoietic stem cell (HSC) gene therapy requires efficient transduction and engraftment of HSCs. However, the HSC transduction remains inefficient in spite of many improved vector systems, and the engraftment is minimal unless toxic marrow conditioning is conducted. To try to overcome these problems all together, we have accomplished two technological innovations. One is a novel transplantation method, bone marrow replacement (BMR), which would enable the engraftment of transplanted cells in bone marrow without marrow conditioning. The other is in vivo expansion of gene-modified cells after transplantation by the selective amplifier gene (SAG). In this study, we have examined the efficacy and safety of the combination of the two methods in nonhuman primates.Cynomolgus CD34+ cells were isolated and retrovirally transduced with an SAG encoding a chimeric protein between the thrombopoietin receptor (c-Mpl) as a growth signal generator and the erythropoietin (EPO) receptor as a molecular switch. The transduced cells were directly injected (autologously) into the femur and humerus bones of which marrow cavity had been gently irrigated with saline prior to the injection (BMR). EPO was administered to the animals after the BMR. As a control, CD34+ cells were genetically marked with the non-expression vector (without SAG) and autologously returned into the animals by BMR or by intravenous injection. All animals did not receive marrow conditioning. After transplantation, in vivo marking levels were monitored.After BMR without SAG, 2–30% of colony-forming cells (CFUs) from the non-implanted marrow were found to be gene-marked over long-term (> 1 year, n = 2). This result indicates that cells implanted by BMR rapidly relocate from the implanted marrow to another (within two weeks post-BMR). The marking levels in the peripheral blood, however, remained very low (
[Show abstract][Hide abstract] ABSTRACT: Although airways are important targets for gene therapy for diseases such as cystic fibrosis or asthma, few vectors can transduce these organs. Recombinant Sendai virus (SeV) vectors can easily penetrate the mucus layer and efficiently transduce airway epithelial cells from apical side. However, the expression of transgene by the vectors of present design is transient and generally lasts only for several days. To improve lentivirus entry into airway epithelial cells, we have attempted to make the novel pseudotyped Simian Immunodeficiency Virus (SIV) vector with SeV envelope proteins, F and HN. The SIV vector could not be pseudotyped with the intact F and HN, but could be successfully pseudotyped after modification of the cytoplasmic portion of both proteins (Kobayashi et al. ASGT 2002). The new F and HN pseudotyped SIV vector was produced to the titers of more than 106 TU/ml and could be easily concentrated by centrifugation. When 108TU of SIV pseudotyped with F and HN was administered to mouse lungs through “sniffing”, distinct transgene (GFP) expression was detected in the alveolar epithelium (Figure 1) and bronchiolar epithelial cells (Figure 2) ten days after the transduction. In contrast, VSV-G pseudotyped SIV vectors did not transduce the lung. These results suggest that our novel pseudotyped SIV vector with F and HN may be of great value as a tool for gene therapy of respiratory diseases.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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 x 10(7) or 2.5 x 10(8) 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.