Correction of chromosomal mutation and random integration in embryonic stem cells with helper-dependent adenoviral vectors

Abstract

For gene therapy of inherited diseases, targeted integration/gene repair through homologous recombination (HR) between exogenous and chromosomal DNA would be an ideal strategy to avoid potentially serious problems of random integration such as cellular transformation and gene silencing. Efficient sequence-specific modification of chromosomes by HR would also advance both biological studies and therapeutic applications of a variety of stem cells. Toward these goals, we developed an improved strategy of adenoviral vector (AdV)-mediated HR and examined its ability to correct an insertional mutation in the hypoxanthine phosphoribosyl transferase (Hprt) locus in male mouse ES cells. The efficiency of HR was compared between four types of AdVs that contained various lengths of homologies at the Hprt locus and with various multiplicities of infections. The frequency of HR with helper-dependent AdVs (HD AdVs) with an 18.6-kb homology reached 0.2% per transduced cell at a multiplicity of infection of 10 genomes per cell. Detection of random integration at DNA levels by PCR revealed extremely high efficiency of 5% per cell. We also isolated and characterized chromosomal sites where HD AdVs integrated in a random manner. In contrast to retroviral, lentiviral, and adeno-associated viral vectors, which tend to integrate into genes, the integration sites of AdV was distributed randomly inside and outside genes. These findings suggest that HR mediated by HD AdVs is efficient and relatively safe and might be a new viable option for ex vivo gene therapy as well as a tool for chromosomal manipulation of a variety of stem cells.

Fig. 1.

Schematic representations of Hprt-targeting AdVs. Locations of Hprt genomic sequence (black box), exons (white box), inverted terminal repeats, and the packaging signal of Ad5 (arrows), adenoviral sequence (striped box),β-geo marker gene, and stuffer DNA fragment (thin line) are shown. The locations of two PCR primer pairs, used to detect HD AdHprt18.6, are shown as PCR 1 and PCR 2.

Fig. 2.

Frequencies of homologous recombination and random integration. Cells were infected with each targeting vector at an moi of 10, 100, and 1,000. Selection was initiated 4 days after infection. The values were determined by dividing the number of G418 or HAT-resistant colonies by the total number of cells plated. Frequencies of random integration and HR are shown in white bars and black bars, respectively. The numbers enclosed in parentheses indicate the percentage of HR in random integration.

Fig. 3.

Homologous recombination in smaller numbers of target cells. AB1/RV7.0PGK cells plated in a 6-well, 24-well, and 96-well at a cell density of 2 × 10⁵ cells per cm² in duplicate. Each cell plate was infected with HD AdHprt18.6 vector at an moi of 10. Two days after infection, the HAT selection was started. Frequency of HR was determined by dividing the total number of HAT-resistant colonies by the total number of cells plated, which were normalized by colony forming efficiency. Experiments were performed in duplicates.