A new generation of human artificial chromosomes for functional genomics and gene therapy

Abstract

Since their description in the late 1990s, human artificial chromosomes (HACs) carrying a functional kinetochore were considered as a promising system for gene delivery and expression with a potential to overcome many problems caused by the use of viral-based gene transfer systems. Indeed, HACs avoid the limited cloning capacity, lack of copy number control and insertional mutagenesis due to integration into host chromosomes that plague viral vectors. Nevertheless, until recently, HACs have not been widely recognized because of uncertainties of their structure and the absence of a unique gene acceptor site. The situation changed a few years ago after engineering of HACs with a single loxP gene adopter site and a defined structure. In this review, we summarize recent progress made in HAC technology and concentrate on details of two of the most advanced HACs, 21HAC generated by truncation of human chromosome 21 and alphoid(tetO)-HAC generated de novo using a synthetic tetO-alphoid DNA array. Multiple potential applications of the HAC vectors are discussed, specifically the unique features of two of the most advanced HAC cloning systems.

Fig. 1

An example of construction of the engineered human artificial chromosome via top-down approach. a The human chromosome 21 was transferred from human cells to recombination-proficient chicken DT40 cells. The HAC was generated by subsequent truncation of the p- and q- arms by targeting constructs in DT40 cells. During arms truncation, a loxP gene loading site was inserted into the HAC in DT40 cells. After the HAC transfer to Chinese hamster ovary (CHO) (hprt −/−) cells, a desired gene can be cloned into the loxP site of the HAC by Cre-loxP-mediated gene insertion. Then, the HAC with a gene of interest can be transferred to desired recipient cells via a micro-cell-mediated transfer technique (MMCT) for gene complementation assays. b A map of the linear 21HAC vector. The TEL/Δq-hisD and TEL/Δp-PGK-Puro constructs were used for chromosome 21 truncation. Two remaining pericentromeric contigs, AP001657 and AL163201, are shown. The 5′HPRT-loxP-Hyg-Tk and pGKneo-FRT-3′Zeo cassettes with loxP and FRT gene loading sites were included in the HAC by homologous recombination. Cre-loxP-mediated gene insertion is accompanied by reconstitution of the HPRT gene. FLP-FRT-mediated gene insertion is accompanied by reconstitution of the function of the Zeo gene. The HAC is marked by the EGFP gene

Fig. 2

Generation of human artificial chromosomes via bottom-up approach. Each human chromosome contains a centromere consisting of identical (high-order repeats or HOR) and diverged alphoid DNA repeats that form an array of 0.5–5 Mb in size. A HOR DNA array of 30–200 kb in size isolated as a BAC forms a HAC after its transfection into human fibrocarcoma HT1080 cells. HAC formation is accompanied by BAC DNA multimerization up to 1–5 Mb. Highly diverged alphoid DNA arrays lacking CENP-B boxes do not form a HAC. Instead, such arrays randomly integrate into human chromosomes

Fig. 3

An example of construction of the de novo generated human artificial chromosome via bottom-up approach using a synthetic alphoid DNA array. a Schematic representation of construction of the synthetic tetO-containing DNA tandem repeats array by rolling circle amplification (RCA) in vitro and transformation-associated recombination (TAR) cloning in yeast cells. The first step included amplification of the dimer by RCA up to 5–10 kb in size fragments. One monomer of the dimer is derived from a chromosome 17 alphoid type I 16-mer unit and contains a CENP-B box. The second monomer is a wholly synthetic sequence derived from alohoid DNA consensus, with sequences corresponding to the CENP-B box replaced by a 42-bp tetO motif. The second step included co-transformation of the RCA-amplified fragments into yeast cells along with a vector containing alphoid-specific hooks. End-to-end recombination of alphoid DNA fragments followed by interaction of the recombined fragments with the vector resulted in the rescue of an approximately 50-kb array as a circular molecule in yeast. The targeting vector contains a yeast cassette, HIS3/CEN/ARS (a selectable marker HIS3, a centromere sequence CEN6 from yeast chromosome VI and yeast origin of replication ARSH4) and a mammalian selectable marker (the Blasticidin resistance gene) and a BAC replicon that allows a YAC clone to be transferred into E. coli cells. b The alphoid^tetO-HAC loss induced by targeting of the transcriptional silencer (tTS) fused with the tet-repressor (tetR) into the HAC kinetochore. After expression of a chromatin modifier gene (tTS), the HAC is maintained stably when cells are growing in the presence of doxycyclin that prevents binding of tTS to tetO sequences or the HAC is destabilized when cells are growing in the absence of doxycyclin

Fig. 4

Insertion of a gene loading site into the alphoid^tetO-HAC. a The alphoid^tetO-HAC was transferred from human HT1080 cells to recombination-proficient chicken DT40 cells. A loxP gene loading site was inserted into the alphoid^tetO-HAC by homologous recombination in DT40 cells. The alphoid^tetO-HAC with the loxP site was transferred to hamster CHO (hprt −/−) cells to insert the desired gene. The alphoid^tetO-HAC with the gene of interest can be transferred to desired recipient cells via microcell-mediated chromosome transfer technique (MMCT). b A scheme of gene loading into the unique loxP gene acceptor site of the alphoid^tetO-HAC. A desired gene can be cloned into the HAC by Cre-loxP mediated gene insertion followed by reconstitution of the HPRT gene

Fig. 5

A scheme of selective gene isolation from human genomic DNA by transformation-associated recombination (TAR-cloning) in the yeast S. cerevisiae. a A high-molecular weight human genomic DNA prepared in agarose plugs or in solution is co-transformed along with the TAR vector into yeast spheroplasts. The TAR vector contains a yeast selectable marker HIS3 for selection of yeast transformants on His-minus medium, a yeast centromere for proper propagation of the vector during cell division and two targeting sequences (hooks) homologous to the 5′ and 3′ ends of the gene of interest. After penetration, the targeting hooks of the vector recombine with the homologous sequences of the gene resulting in a rescue of the desired gene as a circular molecule (YAC) in yeast cells. The gene-positive yeast transformants are selected by PCR using diagnostic primers specific for a gene of interest. b Retrofitting of the circular gene-containing YAC into a BAC/YAC. The retrofitting vector contains a yeast selectable marker URA3, a BAC replicon for propagation in bacterial cells, a chloramphenicol gene (Cm), a loxP gene targeting cassette and two targeting sequences homologous to the TAR vector sequence. Transformation of the linearized retrofitting vector into yeast cells containing the YAC with the gene followed by selection on Ura-minus medium results in a rescue of circular molecules in a YAC/BAC form. The YAC/BAC can be transferred from yeast into E. coli cells by electroporation for further BAC DNA isolation

Fig. 6

Schematic diagram of application of the HAC vector system for gene functional analyses and for the treatment of genetic disorders. a A HAC along with a gene(s) of interest is transferred to desired recipient cells (for example, mouse ES cells or patient-derived human cells deficient for this gene). The HAC containing a gene can be utilized for functional analyses in vitro and in vivo, including a humanized mouse model. For alphoid^tetO-HAC, the phenotypes arising from stable gene expression from the HAC can be reversed when the HAC is eliminated from the cells by inactivating its kinetochore that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. b HAC for gene therapy. A HAC containing a therapeutic gene(s) can be utilized for the treatment of patients with genetic disorders. As a first step, pluripotent stem cells are produced from patient fibroblasts using one of the currently available protocols. Then, a HAC carrying a therapeutic gene is introduced into iPS cells from CHO cells using MMCT. A final step includes transplantation of stem cells into the patient