New Vectors for Gene Delivery: Human and Mouse Artificial Chromosomes

Abstract

Abstract 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.