New Vectors for Gene Delivery: Human and Mouse Artificial Chromosomes

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.

Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette

“…Human artificial chromosomes or HAC-based vectors represent a novel system for gene delivery and expression that has several advantages over previous gene and cell therapy strategies ( 1 – 4 ). All HACs by definition contain a functional centromere and, therefore, replicate and segregate like normal chromosomes in human cells without integration into the host genome.…”

“…HACs can be engineered by ‘top-down’ or ‘bottom-up’ ( de novo formation) approaches ( 1 – 4 , 27 , 28 ). The top-down approach is based on telomere-associated chromosome fragmentation in the homologous recombination-proficient chicken DT40 cell line.…”

“…While the alphoid tetO -HAC vector carrying a single copy of tTS provides a simple way to verify phenotypic changes attributed to expression of genes from the HAC, persistence of the HAC with a silenced gene in cells might want to be avoided under some circumstances. For example, this is specifically required when the HAC vector is used for generation of iPS cells ( 3 , 4 , 41 ).…”

A portable BRCA1-HAC (human artificial chromosome) module for analysis of BRCA1 tumor suppressor function

“…HACs are de novo chromosomes that contain functional centromeres permitting their long-term stable maintenance as single copy mini-chromosomes without integration into the host chromosomes. This minimizes such complications as disruption of endogenous genes ( 15 – 18 and references therein). Moreover, HAC vectors have unlimited cloning capacity allowing them to carry entire genomic loci or potentially groups of loci with all regulatory elements that should faithfully mimic the normal pattern of gene expression.…”

Human Artificial Chromosome with Regulated Centromere: A Tool for Genome and Cancer Studies

“…Since their first description in the late 1990s, Human Artificial Chromosomes (HACs) carrying a functional kinetochore have been considered as a promising system for gene delivery and expression with the potential to overcome several problems caused by the use of viral-based gene transfer systems. − HACs avoid the limited cloning capacity, lack of copy number control, and insertional mutagenesis during integration into host chromosomes that have hampered the use of viral vectors. Although it is not as routine as transfection or infection with a virus, HACs along with the genetic loci can be transferred from one cell to another in the laboratory.…”