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.

Recombinant adeno-associated virus (rAAV)-mediated gene transfer is an attractive approach to the treatment of Duchenne muscular dystrophy (DMD). We investigated the muscle transduction profiles and immune responses associated with the administration of rAAV2 and rAAV8 in normal and canine X-linked muscular dystrophy in Japan (CXMD(J)) dogs. rAAV2 or rAAV8 encoding the lacZ gene was injected into the skeletal muscles of normal dogs. Two weeks after the injection, we detected a larger number of beta-galactosidase-positive fibers in rAAV8-transduced canine skeletal muscle than in rAAV2-transduced muscle. Although immunohistochemical analysis using anti-CD4 and anti-CD8 antibodies revealed less T-cell response to rAAV8 than to rAAV2, beta-galactosidase expression in rAAV8-injected muscle lasted for <4 weeks with intramuscular transduction. Canine bone marrow-derived dendritic cells (DCs) were activated by both rAAV2 and rAAV8, implying that innate immunity might be involved in both cases. Intravenous administration of rAAV8-lacZ into the hind limb in normal dogs and rAAV8-microdystrophin into the hind limb in CXMD(J) dogs resulted in improved transgene expression in the skeletal muscles lasting over a period of 8 weeks, but with a declining trend. The limb perfusion transduction protocol with adequate immune modulation would further enhance the rAAV8-mediated transduction strategy and lead to therapeutic benefits in DMD gene therapy.

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