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.