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

Abstract

Since their description in the late 1990s, Human Artificial Chromosomes (HACs) bearing functional kinetochores have been considered as promising systems for gene delivery and expression. More recently a HAC assembled from a synthetic alphoid DNA array has been exploited in studies of centromeric chromatin and in assessing the impact of different epigenetic modifications on kinetochore structure and function in human cells. This HAC was termed the alphoidtetO-HAC, as the synthetic monomers each contained a tetO sequence in place of the CENP-B box that can be targeted specifically with tetR-fusion proteins. Studies in which the kinetochore chromatin of the alphoidtetO-HAC was specifically modified, revealed that heterochromatin is incompatible with centromere function and that centromeric transcription is important for centromere assembly and maintenance. In addition, the alphoidtetO-HAC was modified to carry large gene inserts that are expressed in target cells under conditions that recapitulate the physiological regulation of endogenous loci. Importantly, the phenotypes arising from stable gene expression can be reversed when cells are “cured” of the HAC by inactivating its kinetochore in proliferating cell populations, a feature that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. AlphoidtetO-HAC-based technology has also been used to develop new drug screening and assessment strategies to manipulate the CIN phenotype in cancer cells. In summary, the alphoidtetO-HAC is proving to be a versatile tool for studying human chromosome transactions and structure as well as for genome and cancer studies.

TAR Cloning: Perspectives for Functional Genomics, Biomedicine, and Biotechnology

“…Despite progress with the CRISPR system, which is regarded as an eminent genome engineering tool in therapeutics,6, 50 we still believe that further progress in functional genomics and gene therapy depends on the availability of both full-length genes and a suitable system for gene delivery and gene expression. The most promising vector system is now human artificial chromosomes (HACs) 61, 62, 63, 64, 65, 66, 67, 68, 69. HACs represent extra chromosomes carrying all of the required components of a functional kinetochore.…”

“…They are stably maintained at a low copy in the host nucleus. Furthermore, they contain no viral genes or proteins and, therefore, should not cause severe immunogenic responses, which have been found to be a serious problem with adenoviral vectors 61, 62, 63, 64, 65, 66, 67, 68, 69. HACs are thus intrinsically safer than traditional viral gene therapy systems.…”

“…Combining engineered human chromosomes with TAR cloning technology can greatly advance gene function studies (Figure 3). 61, 62, 65, 73, 74 Moreover, the combination of large-capacity vectors carrying TAR-isolated genes with stem or progenitor cells establishes a novel platform for gene therapy.

Figure 3Schematic Diagram Illustrating the Possible Spectrum of Applications of the HAC-Based Gene Delivery Vector Carrying a TAR-Isolated Human Gene for Pluripotent Stem Cell-Based Regenerative Medicine and Gene Function Studies(A) A full-length TAR-isolated gene (green box) is loaded into the HAC-based vector via the Cre/loxP-mediated recombination in hypoxanthine phosphoribosyltransferase (HPRT)-deficient Chinese hamster ovary (CHO) cells. (B) The HAC, along with a gene of interest, is then delivered to target cells (for example, mouse embryonic stem cells [ESCs] or patient-derived human cells deficient for this gene) via an improved microcell-mediated chromosome transfer (MMCT) procedure 112, 113, 114.

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Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice

“…The alphoid tetO -HAC features a unique loxP gene acceptor site designed for Cre recombinase-mediated insertion of full-length genes in Chinese hamster ovary (CHO) cells, from which the HAC can be transferred into various recipient cells [22]. All aspects of alphoid tetO -HAC applications in synthetic biology, functional genomics, cancer research, and kinetochore investigation have been summarized in the recent review [23]. The alphoid tetO -HAC still requires further functional investigation, particularly with respect to the improvement of its mitotic stability.…”

Novel screen for anti-cancer drugs that elevate chromosome instability (CIN) using human artificial chromosome (HAC)

“…We have developed a new high-throughput assay for measuring CIN. This quantitative assay for chromosome mis-segregation is based on a non-essential human artificial chromosomes (HAC) with a functional kinetochore constructed in our labs for gene delivery [5]. To adopt this HAC for CIN studies, the EGFP transgene was inserted into the HAC [6].…”