Yasuji Ueda

A Cytoplasmic RNA Vector Derived from Nontransmissible Sendai Virus with Efficient Gene Transfer and Expression

ABSTRACT We have recovered a virion from defective cDNA of Sendai virus (SeV) that is capable of self-replication but incapable of transmissible-virion production. This virion delivers and expresses foreign genes in infected cells, and this is the first report of a gene expression vector derived from a defective viral genome of theParamyxoviridae. First, functional ribonucleoprotein complexes (RNPs) were recovered from SeV cloned cDNA defective in the F (envelope fusion protein) gene, in the presence of plasmids expressing nucleocapsid protein and viral RNA polymerase. Then the RNPs were transfected to the cells inducibly expressing F protein. Virion-like particles thus obtained had a titer of 0.5 × 108 to 1.0 × 108 cell infectious units/ml and contained F-defective RNA genome. This defective vector amplified specifically in an F-expressing packaging cell line in a trypsin-dependent manner but did not spread to F-nonexpressing cells. This vector infected and expressed an enhanced green fluorescent protein reporter gene in various types of animal and human cells, including nondividing cells, with high efficiency. These results suggest that this vector has great potential for use in human gene therapy and vaccine delivery systems.

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.

Simian lentiviral vector-mediated retinal gene transfer of pigment epithelium-derived factor protects retinal degeneration and electrical defect in Royal College of Surgeons rats

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.

Pseudotyped lentivirus vectors derived from simian immunodeficiency virus SIVagm with envelope glycoproteins from paramyxovirus.

ABSTRACT We describe the development of novel lentivirus vectors based on simian immunodeficiency virus from African green monkey (SIVagm) pseudotyped with Sendai virus (SeV) envelope glycoproteins. SeV fusion (F) and hemagglutinin-neuraminidase (HN) proteins were successfully incorporated into the SIVagm-based vector by truncation of the cytoplasmic tail of the F protein and by addition of the cytoplasmic tail of SIVagm transmembrane envelope protein to the N terminus of the HN protein. As with the vesicular stomatitis virus G glycoprotein-pseudotyped vector, the mutant SeV F- and HN-pseudotyped SIVagm vector was able to transduce various types of animal and human cell lines. Furthermore, the vector was able to transduce an enhanced green fluorescent protein reporter gene into polarized epithelial cells of rat trachea from the apical and basolateral sides. Therefore, SeV F- and HN-pseudotyped SIVagm vectors have considerable potential for effective use in gene therapy for various therapies, including respiratory diseases.

Engraftment and tumor formation after allogeneic in utero transplantation of primate embryonic stem cells

Background. To achieve human embryonic stem (ES) cell-based transplantation therapies, allogeneic transplantation models of nonhuman primates would be useful. We have prepared cynomolgus ES cells genetically marked with the green fluorescent protein (GFP). The cells were transplanted into the allogeneic fetus, taking advantage of the fact that the fetus is so immunologically immature as not to induce immune responses to transplanted cells and that fetal tissue compartments are rapidly expanding and thus providing space for the engraftment. Methods. Cynomolgus ES cells were genetically modified to express the GFP gene using a simian immunodeficiency viral vector or electroporation. These cells were transplanted in utero with ultrasound guidance into the cynomolgus fetus in the abdominal cavity (n=2) or liver (n=2) at the end of the first trimester. Three fetuses were delivered 1 month after transplantation, and the other, 3 months after transplantation. Fetal tissues were examined for transplanted cell progeny by quantitative polymerase chain reaction and in situ polymerase chain reaction of the GFP sequence. Results. A fluorescent tumor, obviously derived from transplanted ES cells, was found in the thoracic cavity at 3 months after transplantation in one fetus. However, transplanted cell progeny were also detected (∼1%) without teratomas in multiple fetal tissues. The cells were solitary and indistinguishable from surrounding host cells. Conclusions. Transplanted cynomolgus ES cells can be engrafted in allogeneic fetuses. The cells will, however, form a tumor if they “leak” into an improper space such as the thoracic cavity.

In vivo selective expansion of gene-modified hematopoietic cells in a nonhuman primate model

A major problem limiting hematopoietic stem cell (HSC) gene therapy is the low efficiency of gene transfer into human HSCs using retroviral vectors. Strategies, which would allow in vivo expansion of gene-modified hematopoietic cells, could circumvent the problem. To this end, we developed a selective amplifier gene (SAG) consisting of a chimeric gene composed of the granulocyte colony-stimulating factor (G-CSF) receptor gene and the estrogen receptor gene hormone-binding domain. We have previously demonstrated that primary bone marrow progenitor cells transduced with the SAG could be expanded in response to estrogen in vitro. In the present study, we evaluated the efficacy of the SAG in the setting of a clinically applicable cynomolgus monkey transplantation protocol. Cynomolgus bone marrow CD34+ cells were transduced with retroviral vectors encoding the SAG and reinfused into each myeloablated monkey. Three of the six monkeys that received SAG transduced HSCs showed an increase in the levels of circulating progeny containing the provirus in vivo following administration of estrogen or tamoxifen without any serious adverse effects. In one monkey examined in detail, transduced hematopoietic progenitor cells were increased by several-fold (from 5% to 30%). Retroviral integration site analysis revealed that this observed increase was polyclonal and no outgrowth of a dominant single clonal population was observed. These results demonstrate that the inclusion of our SAG in the retroviral construct allows selective in vivo expansion of genetically modified cells by a non-toxic hormone treatment.

Cotransplantation with MSCs improves engraftment of HSCs after autologous intra-bone marrow transplantation in nonhuman primates

OBJECTIVE Hematopoietic stem cells (HSCs) reside in the osteoblastic niche, which consists of osteoblasts. Mesenchymal stromal cells (MSCs) have an ability to differentiate into osteoblasts. Here, using nonhuman primates, we investigated the effects of cotransplantation with MSCs on the engraftment of HSCs after autologous intra-bone marrow transplantation. MATERIALS AND METHODS From three cynomolgus monkeys, CD34-positive cells (as HSCs) and MSCs were obtained. The former were divided into two equal aliquots and each aliquot was genetically marked with a distinctive retroviral vector to track the in vivo fate. Each HSC aliquot with or without MSCs was autologously injected into the bone marrow (BM) cavity of right or left side, enabling the comparison of in vivo fates of the two HSC grafts in the same body. RESULTS In the three monkeys, CD34(+) cells transplanted with MSCs engrafted 4.4, 6.0, and 1.6 times more efficiently than CD34(+) cells alone, as assessed by BM colony polymerase chain reaction. In addition, virtually all marked cells detected in the peripheral blood were derived from the cotransplantation aliquots. Notably, colony-forming units derived from the cotransplantation aliquots were frequently detected in BM distant sites from the injection site, implying that cotransplantation with MSCs also restored the ability of gene-marked HSCs to migrate and achieve homing in the distant BM. CONCLUSION Cotransplantation with MSCs would improve the efficacy of transplantation of gene-modified HSCs in primates, with enhanced engraftment in BM as well as increased chimerism in peripheral blood through migration and homing.

Sustained and NK/CD4+ T Cell-Dependent Efficient Prevention of Lung Metastasis Induced by Dendritic Cells Harboring Recombinant Sendai Virus

We recently demonstrated efficient antitumor immunity against murine tumors using dendritic cells (DCs) activated by recombinant Sendai viruses (rSeVs), and proposed a new concept, “immunostimulatory virotherapy,” for cancer immunotherapy. However, there has been little information on the efficacy of this method in preventing metastatic diseases. In this study, we investigated the efficacy of vaccinating DCs activated by fusion gene-deleted nontransmissible rSeV (rSeV/dF) using a murine model of lung metastasis. Bolus and i.v. administration of DCs harboring rSeV/dF-expressing GFP without pulsation of tumor Ag (DC-rSeV/dF-GFP) 2 days before tumor inoculation showed efficient prevention against lung metastasis of c1300 neuroblastoma, but not of RM-9 prostatic cancer. We found that the timing of DC therapy was critical for the inhibition of pulmonary metastasis of RM-9, and that the optimal effect of DCs was seen 28 days before tumor inoculation. Interestingly, the antimetastatic effect was sustained for over 3 mo, even when administered DCs were already cleared from the lung and organs related to the immune system. Although NK cell activity had already declined to baseline at the time of tumor inoculation, Ab-mediated depletion studies revealed that CD4+ cells as well as the presence of, but not the activation of, NK cells were crucial to the prevention of lung metastasis. These results are the first demonstration of efficient inhibition of lung metastasis via bolus administration of virally activated DCs that was sustained and NK/CD4+ cell-dependent, and may suggest a potentially new mechanism of DC-based immunotherapy for advanced malignancies.

Generation of optimized and urokinase-targeted oncolytic Sendai virus vectors applicable for various human malignancies

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.

Simian immunodeficiency virus-based lentivirus vector for retinal gene transfer: a preclinical safety study in adult rats

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.