Refined human artificial chromosome vectors for gene therapy and animal transgenesis

• Published in: Gene therapy Vol. 18; no. 4; pp. 384 - 393
• Main Authors: Kazuki, Y, Hoshiya, H, Takiguchi, M, Abe, S, Iida, Y, Osaki, M, Katoh, M, Hiratsuka, M, Shirayoshi, Y, Hiramatsu, K, Ueno, E, Kajitani, N, Yoshino, T, Kazuki, K, Ishihara, C, Takehara, S, Tsuji, S, Ejima, F, Toyoda, A, Sakaki, Y, Larionov, V, Kouprina, N, Oshimura, M
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.04.2011; Nature Publishing Group
• Subjects: 631/1647/2300/1514; 631/61/17/1511; 631/61/201; Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy; Animals; Applied cell therapy and gene therapy; Biological and medical sciences; Biomedical and Life Sciences; Biomedicine; Biotechnology; Cell Biology; Cell Line; Chromosomes; Chromosomes, Artificial, Human; Chromosomes, Human, Pair 21; Cloning, Molecular; Fundamental and applied biological sciences. Psychology; Gene Expression; Gene Therapy; Gene Transfer Techniques; Genetic research; Genetic Therapy - methods; Genetic Vectors; Health aspects; Health. Pharmaceutical industry; Herpes simplex virus; Human Genetics; Humans; Industrial applications and implications. Economical aspects; Medical sciences; Mice; Nanotechnology; Original; original-article; Polymerase chain reaction; Recombination, Genetic; Transfusions. Complications. Transfusion reactions. Cell and gene therapy; Transgenic animals; Japan; human artificial chromosome; animal transgenesis; microcell-mediated chromosome transfer; Human; Animal; Transfer; Gene therapy; Artificial chromosome; Vector
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/gt.2010.147

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.