Induced pluripotent stem cells (iPSC) have been generated from somatic cells by introducing reprogramming factors. Integration of foreign genes into the host genome is a technical hurdle for the clinical application. Here, we show that Sendai virus (SeV), an RNA virus and carries no risk of altering host genome, is an efficient solution for generating safe iPSC. Sendai-viral human iPSC expressed pluripotency genes, showed demethylation characteristic of reprogrammed cells. SeV-derived transgenes were decreased during cell division. Moreover, viruses were able to be easily removed by antibody-mediated negative selection utilizing cell surface marker HN that is expressed on SeV-infected cells. Viral-free iPSC differentiated to mature cells of the three embryonic germ layers in vivo and in vitro including beating cardiomyocytes, neurons, bone and pancreatic cells. Our data demonstrated that highly-efficient, non-integrating SeV-based vector system provides a critical solution for reprogramming somatic cells and will accelerate the clinical application.

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 transgenefree iPSCs from cord blood cells should be an important step in providing allogeneic iPSC-derived therapy in the future.regenerative medicine | nonintegrating RNA vector

The formation of nontransmissible virus-like particles (NTVLP) by cells infected with F-deficient Sendai virus (SeV/⌬F) was found to be temperature sensitive. Analysis by hemagglutination assays and Western blotting demonstrated that the formation of NTVLP at 38°C was about 1/100 of that at 32°C, whereas this temperature-sensitive difference was only moderate in the case of F-possessing wild-type SeV. In order to reduce the NTVLP formation with the aim of improving SeV for use as a vector for gene therapy, amino acid substitutions found in temperature-sensitive mutant SeVs were introduced into the M (G69E, T116A, and A183S) and HN (A262T, G264R, and K461G) proteins of SeV/⌬F to generate SeV/M ts HN ts ⌬F. The use of these mutations allows vector production at low temperature (32°C) and therapeutic use at body temperature (37°C) with diminished NTVLP formation. As expected, the formation of NTVLP by SeV/M ts HN ts ⌬F at 37°C was decreased to about 1/10 of that by SeV/⌬F, whereas the suppression of NTVLP formation did not cause either Sendai virus (SeV) is an enveloped virus with a nonsegmented negative-strand RNA genome and is a member of the family Paramyxoviridae. The SeV genome contains six major genes arranged in tandem. The three virus-derived proteins nucleoprotein (NP), phosphoprotein (P), and large protein (L) form a ribonucleoprotein complex (RNP) with the SeV genomic RNA, and the RNP acts as a template for transcription and replication. Matrix protein (M) is a lining protein of the viral particle and is involved in the assembly of the particle. Two spike proteins, hemagglutinin-neuraminidase (HN) and fusion protein (F), mediate the attachment of virions and penetration of RNPs into infected cells, respectively (18). SeV replication appears to be independent of nuclear functions, and SeV does not have a DNA phase throughout its life cycle, so the virus is unable to transform cells by integrating its genetic information into the cellular genome (18).Since SeV infects and replicates in most mammalian cells, including human cells, and directs high-level expression of the genes it carries, vectors derived from SeV are expected to be useful for gene therapy by expressing therapeutic genes and vaccine antigens (16,21,29,36,38). In particular, we plan the clinical application of an SeV vector carrying human fibroblast growth factor 2 for the treatment of peripheral arterial disease. F-deficient SeV (SeV/⌬F) has thus been produced and demonstrated to be non-self-transmissible due to loss of the F gene in its genomic RNA (19). Recently, we have found that nontransmissible virus-like particles (NTVLP) are formed in cells infected with SeV/⌬F. The NTVLP formation should be reduced for the next-generation SeV vector rather than retained, although so far it has not been proven that NTVLP formation brings about any kind of adverse effect in vivo. In this study, we first characterized the virion formation of SeV/⌬F and showed that loss of the F gene from the SeV genome caused temperature sensitivity of NTVLP formation....

A new recombinant Sendai virus vector (SeV/⌬M), in which the gene encoding matrix (M) protein was deleted, was recovered from cDNA and propagated in a packaging cell line expressing M protein by using a Cre/loxP induction system. The titer of SeV/⌬M carrying the enhanced green fluorescent protein gene in place of the M gene was 7 ؋ 10 7 cell infectious units/ml or more. The new vector showed high levels of infectivity and gene expression, similar to those of wild-type SeV vector, in vitro and in vivo. Virus maturation into a particle was almost completely abolished in cells infected with SeV/⌬M. Instead, SeV/⌬M infection brought about a significant increase of syncytium formation under conditions in which the fusion protein was proteolytically cleaved and activated by trypsin-like protease. This shows that SeV/⌬M spreads markedly to neighboring cells in a cell-to-cell manner, because both hemagglutinin-neuraminidase and active fusion proteins are present at very high levels on the surface of cells infected with SeV/⌬M. Thus, SeV/⌬M is a novel type of vector with the characteristic features of loss of virus particle formation and gain of cell-to-cell spreading via a mechanism dependent on the activation of the fusion protein.

We here report the results of a Phase I/IIa open-label four dose-escalation clinical study assessing the safety, tolerability, and possible therapeutic efficacy of a single intramuscular administration of DVC1-0101, a new gene transfer vector based on a nontransmissible recombinant Sendai virus (rSeV) expressing the human fibroblast growth factor-2 (FGF-2) gene (rSeV/dF-hFGF2), in patients with peripheral arterial disease (PAD). Gene transfer was done in 12 limbs of 12 patients with rest pain, and three of them had ischemic ulcer(s). No cardiovascular or other serious adverse events (SAEs) caused by gene transfer were detected in the patients over a 6-month follow-up. No infectious viral particles, as assessed by hemagglutination activity, were detected in any patient during the study. No representative elevation of proinflammatory cytokines or plasma FGF-2 was seen. Significant and continuous improvements in Rutherford category, absolute claudication distance (ACD), and rest pain were observed (P < 0.05 to 0.01). To the best of our knowledge, this is the first clinical trial of the use of a gene transfer vector based on rSeV. The single intramuscular administration of DVC1-0101 to PAD patients was safe and well tolerated, and resulted in significant improvements of limb function. Larger pivotal studies are warranted as a next step.

Nuclear import of proteins that contain classical nuclear localization signals (NLS) is initiated by importin␣, a protein that recognizes and binds to the NLS in the cytoplasm. In this paper, we have cloned a cDNA for a novel importin ␣ homologue from rice which is in addition to our previously isolated rice importin ␣1a and ␣2, and we have named it rice importin ␣1b. In vitro binding and nuclear import assays using recombinant importin ␣1b protein demonstrate that rice importin ␣1b functions as a component of the NLS-receptor in plant cells. Analysis of the transcript levels for all three rice importin ␣ genes revealed that the genes were not only differentially expressed but that they also responded to darkadaptation in green leaves. Furthermore, we also show that the COP1 protein bears a bipartite-type NLS and its nuclear import is mediated preferentially by the rice importin ␣1b. These data suggest that each of the different rice importin ␣ proteins carry distinct groups of nuclear proteins, such that multiple isoforms of importin ␣ contribute to the regulation of plant nuclear protein transport.The most characteristic feature of an eukaryotic cell is the presence of a nuclear envelope, which separates the cell into two major compartments, the nucleus and the cytoplasm. Communication between these two compartments takes place through the nuclear pore complex (NPC) 1 (for review, see Refs.1-3). The NPC allows molecules smaller than 40 -60 kDa to diffuse across, while larger proteins and RNA-protein complexes must be actively transported through the NPC in a signal-mediated and energy-dependent manner. Nuclear proteins involved in nuclear activities, such as DNA replication, transcriptional RNA synthesis, and RNA splicing, must enter into the nucleus. Conversely, RNA, such as mRNA synthesized in the nucleus, must be transported into the cytoplasm where it is translated to protein. In plants, nucleocytoplasmic transport has been implicated in functional regulation of a number of plant photomorphogenesis related protein factors (4). For example, constitutive photomorphogenic 1 (COP1), a repressor of photomorphogenesis, has been shown to shuttle between the nucleus and cytoplasm in response to a change of light environment. COP1 exists predominantly in the cytoplasm in the light while it accumulates in the nucleus in the dark, suggesting that nuclear protein transport is an underlying mechanism for the regulation of COP1 activity (5). Multiple pathways of nucleocytoplasmic transport have been identified, each likely to be involved in carrying a distinct group of proteins (for review, see Refs. 3 and 6). Among them, the best characterized is the import of proteins containing a classical nuclear localization signal (NLS) that consists of either a short stretch of 3-5 basic amino acids or two basic domains separated by a spacer, referred to as monopartite and bipartite NLS, respectively (7). Yeast mating factor (Mat␣-2) contains a NLS consisting of basic and hydrophobic amino acid residues and has also been shown to...

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Induced pluripotent stem cells (iPSCs) are potentially valuable cell sources for disease models and future therapeutic applications; however, inefficient generation and the presence of integrated transgenes remain as problems limiting their current use. Here, we developed a new Sendai virus vector, TS12KOS, which has improved efficiency, does not integrate into the cellular DNA, and can be easily eliminated. TS12KOS carries KLF4, OCT3/4, and SOX2 in a single vector and can easily generate iPSCs from human blood cells. Using TS12KOS, we established iPSC lines from chimpanzee blood, and used DNA array analysis to show that the global gene-expression pattern of chimpanzee iPSCs is similar to those of human embryonic stem cell and iPSC lines. These results demonstrated that our new vector is useful for generating iPSCs from the blood cells of both human and chimpanzee. In addition, the chimpanzee iPSCs are expected to facilitate unique studies into human physiology and disease.