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Human Artificial Chromosome Gene Therapy (2005) 12, 852–856 & 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00 www.nature.com/gt BRIEF COMMUNICATION Human artificial chromosome (HAC) vector provides long-term therapeutic transgene expression in normal human primary fibroblasts M Kakeda1, M Hiratsuka1,2, K Nagata1, Y Kuroiwa1, M Kakitani1, M Katoh2, M Oshimura2 and K Tomizuka1,2 1Pharmaceutical Research Laboratories, Pharmaceutical Division, Kirin Brewery Co., Ltd, Takasaki-shi, Gunma, Japan; and 2Biomedical Science, Institute of Regenerative Medicine and Bio-function, Graduate School of Medical Science, Tottori University, Nishimachi, Yonago-shi, Tottori, Japan Human artificial chromosomes (HACs) segregating freely the generation of cytogenetically normal hPFs harboring the from host chromosomes are potentially useful to ensure both structurally defined and extra HAC vector. This introduced safety and duration of gene expression in therapeutic gene HAC vector was retained stably in hPFs without translocation delivery. However, low transfer efficiency of intact HACs to of the HAC on host chromosomes. We also achieved the the cells has hampered the studies using normal human long-term production of human erythropoietin for at least 12 primary cells, the major targets for ex vivo gene therapy. To weeks in them. These results revealed the ability of HACs as elucidate the potential of HACs to be vectors for gene novel options to circumvent issues of conventional vectors therapy, we studied the introduction of the HAC vector, which for gene therapy. is reduced in size and devoid of most expressed genes, into Gene Therapy (2005) 12, 852–856. doi:10.1038/ normal primary human fibroblasts (hPFs) with microcell- sj.gt.3302483; Published online 3 March 2005 mediated chromosome transfer (MMCT). We demonstrated Keywords: human artificial chromosome (HAC); microcell-mediated chromosome transfer (MMCT); normal primary fibroblasts; transgene expression; erythropoietin (EPO) Every vector system currently available for gene therapy down), and to assemble a new chromosome de novo has some drawbacks and advantages.1 The use of viral from its constituent DNA elements (bottom-up). Based vectors such as retroviral and lentiviral vectors allow for on our findings that human chromosomes can be utilized the improved duration of therapeutic gene expression as a vector for animal transgenesis,3,4 we have developed by the facile integration of them into the host genome, the methodology to generate top-down HACs by but are plagued by safety concerns such as malignant manipulating human chromosomes with telomere-direc- transformation. On the other hand, nonviral vectors are ted chromosome truncation and homologous recombina- recognized to be nonpathogenic, while less efficient at tion in chicken DT40 cells.5 Using this technology, our introducing and maintaining the transgene expression. group recently constructed a novel human chromosome Artificially engineered human chromosomes are main- 21-derived HAC vector (21DpqHAC vector),6 which was tained independent of the host chromosome through a devoid of most expressed genes by telomere truncation set of cell divisions.2 Accordingly, the host genome is not at the genomic regions proximal to the centromere in disrupted, and the expression of the transgene could be both p (at AL163201 locus) and q (AL163204 locus) arms. sustained for a prolonged period without suffering the A loxP sequence was then introduced into the q arm (at effects of its surrounding sequence on the host genome. AL163203 locus) of the 21DpqHAC vector, which permits These are the ideal properties required for vectors in the site-specific insertion of circular DNA containing the gene therapy, and therefore the utilization of human transgene expression unit by the Cre-loxP system (Figure artificial chromosomes (HACs) could circumvent the 1a). We also showed the microcell-mediated chromo- issues of conventional vectors. some transfer (MMCT)7,8 into an immortalized human There are two basic strategies to construct HAC fibrosarcoma cell line (HT1080) and the mitotic stability vectors:2 to manipulate a natural human chromosome and persistent expression of EGFP gene in the resultant for generating size-reduced minichromosomes (top- HT1080 clones. An autotransplantation of ex vivo transduced normal human primary fibroblasts (hPFs) with the transgene of Correspondence: Dr K Tomizuka, Pharmaceutical Research Laboratories, interest is a potential approach in replacement therapy Pharmaceutical Division, Kirin Brewery Co., Ltd, 3 Miyahara-cho, Takasaki-shi, Gunma 370-1295, Japan for hormone or metabolic enzyme deficiencies, because Received 30 September 2004; accepted 27 December 2004; published the hPFs could be readily obtained from patients and online 3 March 2005 cultured for further genetic modifications. However, the Usefulness of HAC in therapeutic gene delivery M Kakeda et al 853 hPFs carrying the HAC vector, we also showed the long-term expression of therapeutic transgene, human erythropoietin (EPO) as a model, which is a growth factor for erythroid cells and widely used for the clinical treatment of anemia in renal insufficiency.11 Although the introduction of HACs derived from human chromosome 14 fragment (SC20) into normal bovine primary fibroblasts (bPFs) with MMCT was shown for preparing the donor cells of the somatic nuclear transfer to generate trans-chromosomic calves,12 the retention and structure of the HACs in the transduced bPFs after a set of cell divisions remain to be elucidated because of the low transfer efficiency (approximately 1 to 10 Â 10À6 in immortalized cells)7 in MMCT and the limited cell division capacity of bPFs . To investigate the feasibility of minichromosome transfer into normal hPFs by MMCT, we employed HFL-1 cells (RCB0521, RIKEN, Tsukuba, Japan) as recipient hPFs and first examined the SC20 fragment, which contains neor gene and was shown to be stable in various human and mouse cells.13 It was under the condition of 45% polyethylene glycol 1500 containing 10% dimethyl sulfoxide in DMEM that the most frequently G418- resistant colonies were obtained (about 1.26 Â 10À4 of the transduced cells), indicating that the neor marker on the Figure 1 Construction of the EPO-21DpqHAC vector. (a) Schematic SC20 fragment could be transferred into normal hPFs diagram of the construction of the 21DpqHAC vector.6 The distal q arm and this efficiency was better when compared with that was deleted from human chromosome 21 by telomere-directed truncation at in immortalized cells.7 In addition to HFL-1 cells, the AL163204 locus. For the site-directed insertion of the transgene of interest, SC20 fragment was also successfully transferred into a loxP sequence was introduced at AL163203 locus on the q arm. And the other normal hPFs, HUC-F2 cells and HF19 cells then, the distal p arm was further deleted at AL163201 locus as with the q arm deletion. Triangle indicates artificially synthesized telomere sequence (RCB0436, RCB0210, RIKEN, Tsukuba, Japan, data not for telomere truncation. loxP, loxP sequence. (b) Schematic diagram of the shown), suggesting that MMCT of minichromosomes introduction of the epo expression cassette into the loxP site on the could be applicable to a variety of hPFs from patients as 21DpqHAC vector. CMV, CMV promoter; epo, human EPO cDNA, neo, well as above hPFs. neomycin-resistant gene; and bsr, blasticidine-S-resistant gene. pLN1- We then constructed the expression cassette of human EPO was constructed as follows: SV40 polyA sequence was amplified with epo gene, which is under the control of the CMV primer pairs, 50-CGGGATCCCTCGAGCGAGACATGATAAGATACA TTGATG-30 and 50-GGAAGATCTTCCTAATCAGCCATACCACATTT promoter and SV40 polyA signal, as an application GTAGAGG-30, and pSTneoB25 as a template DNA, using KOD-PLUS example, and introduced them into the 21DpqHAC DNA polymerase (Toyobo, Osaka, Japan). This DNA fragment was vector by Cre-loxP recombination in CHO (#21)–hyg4 digested with BamHI and BglII, and then cloned into the BamHI site of cells6 (EPO-21DpqHAC vector, Figure 1b). We obtained pBS226 (Gibco BRL, Grand Island, NY) as for pBS226-pA. CMV 21 G418-resistant clones (named H4E cells), and all of promoter sequence was amplified with primer pairs, 50-CGGAATTCC 0 0 which secreted EPO into conditioned media (250– GGACATTGATTATTGACTAGTTATTAATAG-3 and 5 -CGGGATCCC 6 GGGTGTCTTCTATGGAGGTCAAAACAG-30, and pBS226 as a tem- 520 IU/10 cells/6 days). The retention of markers for plate DNA, and then cloned into EcoRI–BamHI site of pBS226-pA as for human chromosome 21 (D21S275, PRED65, PRED3) on pLN1. For pLN1-EPO, human EPO coding sequence was amplified with the EPO-21DpqHAC vector was confirmed by PCR. primer pairs, 50-CGGGATCCCGGCCACCATGGGGGTGCACGAAT To examine the structure and copy number of the GTC-30 and 50-CGCTCGAGCGCTATCTGTCCCCTGTCCTGCAGG-30, EPO-21DpqHAC vector in H4E cells, fluorescence in situ and human epo cDNA as a template DNA, and then cloned into BamHI– hybridization (FISH) analysis was carried out on four XhoI site of pLN1. Arrows with short-dashed line indicate XbaI digestion site for Southern blot analysis. Arrows indicate the primer positions for clones using human Cot1 DNA as the probe. In all four M13RV, SVNp1 and NeoRp2. clones, a single spot signal was observed in most of the metaphase specimens (93–99%, n ¼ 50 on each clone), indicating that the EPO-21DpqHAC vector was retained ability of 21DpqHAC as a vector for ex vivo therapeutic as a single copy per cell. No insertion or translocation or gene delivery in human primary cells has
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