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.

Figure 1

Construction of 21HAC1 from hChr.21. (a) Strategy for construction of 21HAC1 from hChr.21. (b–e) FISH analyses for DT40 (hChr.21) (b), DT40 (hChr.21-loxP) (c), DT40 (hChr.21-loxPΔp) (d) and DT40 (hChr.21-loxPΔpq/21HAC1) (e). Digoxigenin-labeled human COT-1 DNA (red) was used to detect the HAC. Biotin-labeled PGK-Hyg, PGK-Puro and β-actin-HisD (green) were used to detect the marker gene on the HAC in c, d and e, respectively. Chromosomal DNA was counterstained with 4,6-diamidino-2-phenylindole (DAPI). The insets show an enlarged image of the modified hChr.21 (HAC) (arrow). (f) Map of the 21HAC1 vector.

Figure 2

Schematic diagram of cloning genes into the 21HAC vector. (a) Homologous recombination-type cloning (sequential gene insertion). The desired gene can be sequentially cloned into a specific site on the HAC in DT40 cells by homologous recombination. The 21HAC2 can be transferred to the hiMSC, mES and HT1080 for each experiment via CHO (hprt−/−)(21HAC2) cells. (b) Insertion-type cloning-1 (HPRT version). The 21HAC1 can be transferred to CHO (hprt−/−) cells. A circular vector can be cloned into the HAC in CHO (hprt−/−) cells by Cre-loxP-mediated gene insertion with reconstitution of the HPRT gene. (c) Insertion-type cloning-2 (neo version). The 21HAC4 modified from 21HAC1 can be transferred to CHO-K1 cells. A circular vector can be cloned into the HAC in CHO-K1 cells by Cre-loxP-mediated gene insertion with reconstitution of the neo gene.

Figure 3

Detailed map of 21HACs. (a–f) shows homologous regions on hChr.21 for the targeting. The targeting vector, I-EGFP-I-Bsd, DsRed-neo and 3′neo-loxP-Bsd-TK were inserted into the 21HAC1, 21HAC2 and 21HAC1, respectively. The information of the targeting vectors was described in Supplementary Figure S1–S4. The hook for the TAR cloning vector is described in the loci of the 21HAC4.

Figure 4

Analysis of a HAC containing desired genes. (a–f) FISH analyses for DT40 (21HAC2) (a), DT40 (21HAC3) (b), CHO (21HAC1) (c), CHO (GFP-21HAC1) (d), CHO (21HAC4) (e) and CHO (HPRT-21HAC4) (f). Digoxigenin-labeled human COT-1 DNA (red) was used to detect the HAC. Biotin-labeled CAG–EGFP, CMV-DsRed and RP6-127C8 (green) were used to detect the marker gene on the HAC in (a and d), (b) and (f), respectively. Chromosomal DNA was counterstained with 4,6-diamidino-2-phenylindole (DAPI). The inset shows an enlarged image of the HAC (arrow). (g) Expression of the fluorescent gene on the HAC in DT40 cells. Phase-contrast (top panel), GFP-fluorescence (middle panel) and DsRed-fluorescence (bottom panel) micrographs are shown. (h) Expression of the fluorescent gene on the 21HAC1 in CHO cells. (i) Representative genomic PCR and reverse transcriptase (RT)-PCR data for detecting HPRT-21HAC4 in CHO cells. HT1080 and CHO (21HAC4) cells were used as positive and negative controls, respectively. β-actin was used as an internal control.

Figure 5

Stability of 21HAC2 in vitro and in vivo. (a) Morphology of hiMSC (21HAC2) cells. Phase-contrast (top panel) and fluorescence (bottom panel) micrographs are shown. (b) FISH analyses for hiMSC (21HAC2) cells. An arrow indicates the 21HAC2 and the inset shows an enlarged image of the 21HAC2. Digoxigenin-labeled p11-4, derived from hChr.21 (red) was used to detect the HAC. (c) Mitotic stability of 21HAC2 in hiMSC (21HAC2) cells as a function of population doubling (PDL). (d) Morphology of E14 (21HAC2) cells. Phase-contrast (top panel) and fluorescence (bottom panel) micrographs are shown. (e) GFP expression in various tissues of trans-chromosomic (Tc) mouse containing 21HAC2. Bright (left panel) and fluorescence (right panel) micrographs are shown. (f) FISH analyses for Tc tail-fibroblasts containing the 21HAC2. Digoxigenin-labeled human COT-1 DNA (red) was used to detect the HAC. An arrow indicates the 21HAC2 and the inset shows an enlarged image of the 21HAC2.

Figure 6

Effect of GCV on HT1080 (21HAC2) with HSV-TK in vitro and in vivo. (a) Morphology of HT1080 (21HAC2) cells. Phase-contrast (top panel) and fluorescence (bottom panel) micrographs are shown. (b) FISH analyses for HT1080 (21HAC2) cells. An arrow indicates the 21HAC2 and the inset shows an enlarged image of the 21HAC2. Digoxigenin-labeled p11-4, derived from hChr.21 (red), was used to detect the HAC. (c and d) Growth curve of HT1080 and HT1080 (21HAC2) cells with d or without c GCV selection in vitro. (e) Tumors derived from HT1080 and HT1080 (21HAC2) cells with (left) or without (right) GCV treatment. (f) Tumor weight in HT1080- and HT1080 (21HAC2)-derived tissues with phosphate-buffered saline (PBS) or GCV treatment. A statistical analysis was performed using a two-tailed Student's t-test.