Human artificial chromosome (HAC) has several advantages as a gene therapy vector, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including the regulatory elements. Induced pluripotent stem (iPS) cells have great potential for gene therapy, as such cells can be generated from the individual's own tissues, and when reintroduced can contribute to the specialized function of any tissue. As a proof of concept, we show herein the complete correction of a genetic deficiency in iPS cells derived from Duchenne muscular dystrophy (DMD) model (mdx) mice and a human DMD patient using a HAC with a complete genomic dystrophin sequence (DYS-HAC). Deletion or mutation of dystrophin in iPS cells was corrected by transferring the DYS-HAC via microcell-mediated chromosome transfer (MMCT). DMD patient- and mdx-specific iPS cells with the DYS-HAC gave rise to differentiation of three germ layers in the teratoma, and human dystrophin expression was detected in muscle-like tissues. Furthermore, chimeric mice from mdx-iPS (DYS-HAC) cells were produced and DYS-HAC was detected in all tissues examined, with tissue-specific expression of dystrophin. Therefore, the combination of patient-specific iPS cells and HAC-containing defective genes represents a powerful tool for gene and cell therapies.

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis.

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SUMMARYIn order to determine whether or not IFN-gR is associated with regulatory mechanisms on human eosinophil function, we examined the expression of functional IFN-gR on human peripheral eosinophils. In this study, peripheral blood eosinophils were obtained from seven normal controls and 12 patients (bronchial asthma, n ¼ 9, and hypereosinophilic syndrome (HES), n ¼ 3), and the purity of eosinophils was 97·11 Ϯ 2. 31%, n ¼ 19. We first showed that anti-IFN-gR a-chain MoAb reacted with all tested eosinophils of both normal controls and patients by flow cytometry analysis. We also showed expression of mRNA for the a-chain of IFN-gR in all purified eosinophils of six individuals. Further, to characterize IFN-gR on eosinophils, we did binding experiments with 125 I-IFN-g on purified peripheral eosinophils. The linear Scatchard plot indicated a single type of high-affinity binding sites (dissociation constant (Kd) ¼ 3·89-4·95 × 10 -10 M, numbers of binding sites ¼ 183-233/cell, n ¼ 3). To determine whether IFN-gR on eosinophils is functional, we examined surface eosinophilic cationic protein (ECP) and CD69 induction after IFN-gR ligation with recombinant human IFN-g (rhIFN-g) on eosinophils by flow cytometry. rhIFN-g stimulation significantly induced both ECP and CD69 expression on the 2-18 h-cultured eosinophils in a dose-dependent manner. Further, the effects of rhIFN-g stimulation were significantly blocked by both a neutralizing anti-IFN-g MoAb and a blocking anti-IFN-gR MoAb. These results suggest that human peripheral eosinophils express functional IFN-gR.

The purpose of this study was to determine whether the JAK pathway is involved in eosinophil activation and survival through IFN-gamma receptor signalling in human peripheral eosinophils. Eosinophils were purified from the blood of six atopic disease patients by anti-CD16 magnetic bead-negative selection. IFN-gamma significantly up-regulated survival and CD69 expression in 24-48 h cultured eosinophils. Further, IFN-gamma induced tyrosine phosphorylation of JAK2 in eosinophils, as indicated by Western blot analysis. Finally, the specific JAK2 inhibitor AG-490 inhibited the tyrosine phosphorylation of JAK2, IFN-gamma-induced survival and CD69 expression in eosinophils. In conclusion, these results indicate that IFN-gamma induces eosinophil survival and CD69 expression through the activation of JAK2 in peripheral eosinophils, suggesting that JAK2 may play a significant role in eosinophil regulation by IFN-gamma-IFN-gammaR interaction.