A highly Stable and Nonintegrated Human Artificial Chromosome (HAC) Containing the 2.4 Mb Entire Human Dystrophin Gene

Hoshiya, Hidetoshi; Kazuki, Yasuhiro; Abe, Satoshi; Takiguchi, Masato; Kajitani, Naoto; Watanabe, Yoshinori; Yoshino, T; Shirayoshi, Yasuaki; Higaki, Kazutaka; Messina, Graziella; Cossu, Giulio; Oshimura, Mitsuo

doi:10.1038/mt.2008.253

Cited by 100 publications

(109 citation statements)

References 41 publications

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“…5 CHO cells containing the FVIII-HAC were used as donor microcell hybrids. Briefly, hiMSC cells were fused with microcells prepared from donor hybrid CHO cells that contained FVIII-HAC, and hiMSC cells containing a FVIII-HAC were selected with BS (4 mg ml À1 ).…”

Section: Microcell-mediated Chromosome Transfer and Transfectionmentioning

confidence: 99%

“…The use of currently available viral vector systems may be complemented by the use of human artificial chromosomes (HAC) because HAC replicates and segregates independently from host chromosomes as a minichromosome. [2][3][4][5][6] HAC can be transferred into different cell lines by microcell-mediated chromosome transfer. A HAC vector derived from human chromosome 21 and devoid of all endogenous genes is currently available.…”

Section: Introductionmentioning

confidence: 99%

“…In addition, because HAC persists as a minichromosome, many complications associated with current vectors, including silencing of the therapeutic gene or oncogenesis resulting from integration in host chromosomes, may be minimized or eliminated. Thus, HAC has been offered as an alternative vector in cell-mediated gene therapy, and therapeutic genes have been transferred into multiple stem cell types via HAC, for example, genomic human p53 into multipotent germline stem cell 7 and genomic Duchenne muscular dystrophy into mouse embryonic stem cell 5 and human and mouse induced pluripotent stem cells. 6 HACs support long-term correction of defective genes because expression from and transmission of these vectors are stable throughout many cell divisions in human cells.…”

Section: Introductionmentioning

confidence: 99%

“…6 HACs support long-term correction of defective genes because expression from and transmission of these vectors are stable throughout many cell divisions in human cells. [4][5][6][7][8] This long-term stability is a significant improvement over the stability associated with current gene delivery methods.…”

Section: Introductionmentioning

confidence: 99%

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Integration-free and stable expression of FVIII using a human artificial chromosome

et al. 2011

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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. To examine the copy number effect on the gene expression levels and its stability for a long-term culture for a future application in gene therapy, we constructed a HAC vector carrying the human factor VIII (FVIII) complementary DNA, FVIII-HAC in Chinese hamster ovary (CHO) cells. One and more copies of FVIII gene on the HAC were expressed in the copy-number-dependent manner in the CHO cells. The HAC with 16 copies of FVIII, FVIII (16)-HAC, was transferred from CHO hybrids into a human immortalized mesenchymal stem cell using microcell-mediated chromosome transfer. The expression levels of HAC-derived FVIII transgene products were compared with transfected FVIII plasmids. The former showed expression levels consistent with those of the original clones, even after 50 population doublings, whereas the latter showed a remarkable decrease in expression despite unvarying DNA content, indicating that the gene on the HAC is resistant to gene silencing. These results suggest that the HAC-mediated therapeutic gene-expression system may be a powerful tool for stable expression of transgenes, and possibly for industrial production of gene products.

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Section: Microcell-mediated Chromosome Transfer and Transfectionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Integration-free and stable expression of FVIII using a human artificial chromosome

et al. 2011

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“…We have previously reported the construction of DYS-HAC, which contained a complete 2.4 Mbp genomic dystrophin region including the region that regulates physiological functions, for use in gene therapy Hoshiya et al 2009). The DYS-HAC was transferred to fibroblasts derived from a patient with Duchenne Muscular Dystrophy, and the cells were induced to become induced pluripotent stem cells (iPSCs).…”

mentioning

confidence: 99%

The transfer of human artificial chromosomes via cryopreserved microcells

Uno

Zatti

et al. 2013

Cytotechnology

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Microcell-mediated chromosome transfer (MMCT) technology enables a single and intact mammalian chromosome or megabase-sized chromosome fragments to be transferred from donor to recipient cells. The conventional MMCT method is performed immediately after the purification of microcells. The timing of the isolation of microcells and the preparation of recipient cells is very important. Thus, ready-made microcells can improve and simplify the process of MMCT. Here, we established a cryopreservation method to store microcells at -80°C, and compared these cells with conventionally-(immediately-) prepared cells with respect to the efficiency of MMCT and the stability of a human artificial chromosome (HAC) transferred to human HT1080 cells. The HAC transfer in microcell hybrids was confirmed by FISH analysis. There was no significant difference between the two methods regarding chromosome transfer efficiency and the retention rate of HAC. Thus, cryopreservation of ready-to-use microcells provides an improved and simplified protocol for MMCT.

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New Vectors for Gene Delivery: Human and Mouse Artificial Chromosomes

Oshimura

Kazuki

Iida

et al. 2013

Encyclopedia of Life Sciences

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Human artificial chromosomes (HACs) have been generated mainly by either a ‘top‐down approach’ (engineered creation) or a ‘bottom‐up approach’ ( de novo creation). HACs with acceptor sites exhibit several characteristics required by an ideal gene delivery vector, including stable episomal maintenance and capacity to carry large genomic loci plus their regulatory elements, thus allowing the physiological regulation of the introduced gene in a manner similar to that of native chromosomes. Mouse artificial chromosomes (MACs) with acceptor sites were also created from a native mouse chromosome. The microcell‐mediated chromosome transfer (MMCT) technique for manipulating HACs and MACs in donor cells in order to deliver them to recipient cells is required for each approach. This review describes the lessons learned and prospects identified from studies on the construction of HACs and MACs and their ability to drive exogenous gene expression in cultured cells and transgenic animals via MMCT. The recent emergence of stem cell‐based tissue engineering has opened up new avenues for gene and cell therapies, and possible applications for medical use of HACs and MACs are also proposed. Key Concepts: The microcell‐mediated chromosome transfer method can transfer an intact chromosome or chromosome fragment from donor cell to recipient cell. Human artificial chromosome (HAC), which is an episomal vector derived from a native chromosome, has several advantages: it can contain the entire genome sequences of interest including its regulatory regions with long‐term stable maintenance and gene expression in human cells. The de novo HAC (bottom‐up approach) has been developed with alphoid DNA in HT1080 cells under the condition with lower H3K9me3 methylated alphoid DNA concerning cell‐type‐specific barrier for de novo CENP‐A assembly. The tet‐O HAC is a kind of de novo HAC with a conditional centromere which is constructed with alphoid DNA, and tetracycline operator sequence can be inactivated by expression of tet‐repressor fusion protein for loss of the tet‐O HAC. Mouse artificial chromosome (MAC) is a useful tool for the animal transgenesis because it can be maintained stably in mouse cells and transchromosomic mice through germline transmission. HAC/MAC have a loxP site and/or five recombination platforms to insert circular vectors like bacterial artificial chromosome or make a translocation of the targeting region from other chromosomes. HACs could be used for the complete correction of iPS cells from a patient with Duchenne muscular dystrophy. An HAC which contains Klf4, Sox2, Oct4, c‐Myc and siRNA expression against p53 could reprogramme mouse embryonic fibroblast to pluripotent stem cells. Humanised model mice using human artificial chromosomes, which contain the human CYP3A cluster and the human immunoglobulins including whole 700kb or 1Mb genomic region, have been produced. HACs/MACs might be used for a wide variety of purposes including medical and pharmaceutical applications and even for synthetic biology in sophisticated control.

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A highly Stable and Nonintegrated Human Artificial Chromosome (HAC) Containing the 2.4 Mb Entire Human Dystrophin Gene

Cited by 100 publications

References 41 publications

Integration-free and stable expression of FVIII using a human artificial chromosome

Integration-free and stable expression of FVIII using a human artificial chromosome

The transfer of human artificial chromosomes via cryopreserved microcells

New Vectors for Gene Delivery: Human and Mouse Artificial Chromosomes

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