Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors.

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.

Toward gene therapy for cystic fibrosis using a lentivirus pseudotyped with Sendai virus envelopes.

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.

Efficient expression of a transgene in platelets using simian immunodeficiency virus-based vector harboring glycoprotein Ibalpha promoter: in vivo model for platelet-targeting gene therapy.

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.

Pigment epithelium-derived factor gene therapy targeting retinal ganglion cell injuries: neuroprotection against loss of function in two animal models.

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.

Stable retinal gene expression in nonhuman primates via subretinal injection of SIVagm-based lentiviral vectors.

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.

Acute toxicity study of a simian immunodeficiency virus-based lentiviral vector for retinal gene transfer in nonhuman primates.

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.

A useful approach for the screening of active neural-inducing factors.

The present study suggests that the membrane-binding molecules of mesodermal cells and/or the modulated extracellular matrix (ECM) with them play an important role in induction of the central nervous system. Artificially mesodermalized ectoderm (mE) or chordamesoderm (cM) was placed on a collagen and flbronectin (CF)-coated dish for 24 h. After mechanical removal of the mesoderm sheet, competent ectoderm of early gastrulae was placed on the same spot. Many melanocytes and neuronal cells were observed after 1 week, along with many cells which reacted specifically with a neuralspecific monoclonal antibody. However, when presumptive ectoderm (pE) instead of mE or cM was used as the control, only epidermal cells with cilia were observed in the competent ectoderm, except for a few melanocytes in rare cases. The proteins synthesized and remaining on the CF substrate during placement of the mE and pE were analysed by two-dimensional polyacrylamide gel electrophoresis (PAGE) fluorography. The fluorography indicated that there were significant differences between the polypeptides spots of mE and pE.

pag gene-like protein (ABP-25) of the Cynops embryo: regional distribution and gene expression during early embryogenesis.

A maternal protein showing a unique distribution during early Cynops embryogenesis was screened by monoclonal antibody. The antigen protein, designated as ABP-25 (animal blastomere protein, molecular weight 25,000), was distributed uniformly in the uncleaved egg and concentrated into blastomeres of the animal half during cleavage. At the blastula stage, ABP-25 was definitely localized in cells of the animal half and a polarized distribution was observed within the cytoplasm. During gastrulation, immunohistochemical analysis indicated that the reactivity of the marginal zone (presumptive mesoderm) to the monoclonal antibody ABP-25 decreased after involution. At the end of gastrulation, a polarized distribution was still clearly observed in the ventral epidermis, but not in the neuroectoderm. Both Western and Northern blots indicated that the amount of antigen protein and the intensity of gene expresion were almost constant until the neurula stage. The deduced amino acid sequence of the ABP-25 cDNA showed a strong homology (84%) with that of the pag gene associated with cell proliferation.

High-level in vivo gene marking after gene-modified autologous hematopoietic stem cell transplantation without marrow conditioning in nonhuman primates.

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.

Sustained transgene expression by human cord blood derived CD34+ cells transduced with simian immunodeficiency virus agmTYO1-based vectors carrying the human coagulation factor VIII gene in NOD/SCID mice.

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.