title: A new chromosome 14-based human artificial chromosome (HAC) vector system for efficient transgene expression in human primary cells.
abstract: The use of non-integrating human artificial chromosomes (HACs) in gene therapy possibly allows for safe and reliable genetic modification of human cells without insertional mutagenesis and/or unexpected oncogene activations. Although we previously demonstrated that the HAC provides long-term therapeutic erythropoietin (EPO) production in normal human primary fibroblasts (hPFs), the expression level of EPO was too low to provide medical benefits for human therapy. Thus, the next challenge for the application of this system in therapeutic purposes is to improve the transgene expression on HACs. Here, we newly constructed chromosome 14-based HACs and examined the effects of the telomere and promoter regions on the expression level of the tansgene in hPFs. We showed that the use of natural telomere/sub-telomere and enhancers within the 5' untranslated region of the human ubiquitin C gene greatly increased (over 1000-fold) the EPO production in hPFs. Furthermore, we demonstrated the reprogramming of mouse embryonic fibroblasts by HAC-mediated introduction of four transcription factors, and established induced pluripotent stem cells with no trace of the HACs carrying multiple expression cassettes with large genome fragments. These results indicate that this HAC system could allow us to manipulate multiple transgenes efficiently in human primary cells, providing a promising tool not only for gene therapy but also for investigating genome functions in drug discoveries.
authors: Innovative Drug Research Laboratories, Research Division, Kyowa Hakko Kirin Co., Ltd., 3-6-6 Asahimachi, Machida, Tokyo 194-8533, Japan. minoru.kakeda@kyowa-kirin.co.jp
authors: Minoru
authors: M
authors: Kakeda
authors: Keiko
authors: K
authors: Nagata
authors: Kanako
authors: K
authors: Osawa
authors: Hiroki
authors: H
authors: Matsuno
authors: Masaharu
authors: M
authors: Hiratsuka
authors: Akiko
authors: A
authors: Sano
authors: Akiyo
authors: A
authors: Okazaki
authors: Shingo
authors: S
authors: Shitara
authors: Satoshi
authors: S
authors: Nishikawa
authors: Anri
authors: A
authors: Masuya
authors: Toshihiro
authors: T
authors: Hata
authors: Shun
authors: S
authors: Wako
authors: Mitsuhiko
authors: M
authors: Osaki
authors: Yasuhiro
authors: Y
authors: Kazuki
authors: Mitsuo
authors: M
authors: Oshimura
authors: Kazuma
authors: K
authors: Tomizuka
mesh_terms: D000818:Animals; D002460:Cell Line; D065150:Cellular Reprogramming; D022222:Chromosomes, Artificial, Human / Q000235:genetics*; D002883:Chromosomes, Human, Pair 14 / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D004921:Erythropoietin / Q000235:genetics; D005347:Fibroblasts; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics; D016615:Telomere / Q000235:genetics; D019076:Transgenes
chemical_list: D004921:Erythropoietin