Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology

Abstract

De novo artificial chromosomes expressing genes have been generated in human embryonic stem cells (hESc) and are maintained following differentiation into other cell types. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. De novo HAC are generated following delivery of alpha satellite DNA into target cells. HAC are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation. They are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long-term expression studies in cells unlike other viral gene delivery vectors currently used. Transferring the essential DNA sequences for HAC formation intact across the cell membrane has been challenging for a number of years. A highly efficient delivery system based on HSV-1 amplicons has been used to target DNA directly to the ES cell nucleus and HAC stably generated in human embryonic stem cells (hESc) at high frequency. HAC were detected using an improved protocol for hESc chromosome harvesting, which consistently produced high-quality metaphase spreads that could routinely detect HAC in hESc. In tumour cells, the input DNA often integrated in the host chromosomes, but in the host ES genome, it remained intact. The hESc containing the HAC formed embryoid bodies, generated teratoma in mice, and differentiated into neuronal cells where the HAC were maintained. The HAC structure and chromatin composition was similar to the endogenous hESc chromosomes. This review will discuss the technological advances in HAC vector delivery using HSV-1 amplicons and the improvements in the identification of de novo HAC in hESc.

Chromosome therapy: Potential strategies for the correction of severe chromosome aberrations

“…This would allow supplementation of stable, functional copies of the deleted genes along with the regulatory regions needed to ensure the genes are expressed in the typical physiological spatial and temporal manner for each cell type receiving a copy. HACs have been shown to form functional kinetochores, which allow them to be stable through multiple cellular divisions, and even through differentiation of human embryonic stem cells to multiple cell lineages [Moralli and Monaco, ]. The limitations to this technology are its ability to be delivered to different cell types as the HACs are rapidly degraded when extracellular, thus delivery relies on microcell mediated transfer from one live carrier cell to another.…”

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.…”

Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology

“…Existing strategies for HAC construction can be divided into two classes: top down, based on the truncation of an existing chromosome into a much smaller mini-chromosome suitable for further manipulation, and bottom up, based on transfection of large blocks of centromeric alphoid DNA into human cells with resulting HAC formation (Oshimura et al 2015; Katona 2015; Moralli and Monaco 2015). TAR cloning allows for the construction of synthetic arrays of alphoid DNA with a predetermined structure, which may be then used as substrates for HAC formation (Ebersole et al 2005).…”