Refined human artificial chromosome vectors for gene therapy and animal transgenesis

Abstract

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis.

Stable maintenance of de novo assembled human artificial chromosomes in embryonic stem cells and their differentiated progeny in mice

“…The most advanced HAC of this type (21HAC) was generated by truncation of human chromosome 21. 4 The 21HAC, presumably free of any known genes, has been further modified to contain multiple recombination sites to facilitate loading of sequences of interest. 5 Importantly, this HAC has demonstrated its utility as a high capacity gene therapy vector in mouse models of muscular dystrophies.…”

Integration-free and stable expression of FVIII using a human artificial chromosome

“…Hypoxanthine phosphoribosyl transferase (HPRT)-deficient Chinese hamster ovary (CHO; JCRB0218) hybrids containing the 21HAC2 vector were constructed previously 9 and maintained in Ham's F-12 nutrient mixture (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum and 8 mg ml À1 blasticidin S (BS; Funakoshi, Tokyo, Japan). A hiMSC 10 was maintained in Dulbecco's modified Eagle's medium (Sigma-Aldrich Inc., St Louis, MO, USA) supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS, USA) .…”

“…Each modified pPAC4 plasmid with the FVIII-expression cassettes, abbreviated as FVIII-PAC, had a 3¢ HPRT-loxP site, allowing the FVIII cassettes to transfer readily into the 21HAC2 vector. 9 The 21HAC2 was stably maintained in the CHO clone in selective medium. The CHO cells with a 21HAC2 were co-transfected with 10 mg of the modified pPAC4 vectors carrying the FVIII multicopy cassettes and 1 mg of the Cre-expression vector, pBS185 (Invitrogen) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol.…”

Combinations of chromosome transfer and genome editing for the development of cell/animal models of human disease and humanized animal models

“…In system (a), gene loading with unique sequences in HACs/ MACs via HR in DT40 cells has previously been performed [49,54] (Fig. 3a).…”

“…One such HAC vector (tet-O HAC) with an artificial centromere sequence is conditionally removable from host cells, so is particularly useful for gene function analysis [46]. The top-down approach generates HACs from a native chromosome by chromosome engineering technology, including gene-targeting and telomereassociated chromosome truncation in DT40 cells [47][48][49] (Fig. 1b).…”