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

Abstract

For gene therapy of inherited diseases, targeted integration͞gene 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 helperdependent 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.gene therapy ͉ homologous recombination ͉ hypoxanthine phosphoribosyl transferase

Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors

“…Although the chromosomal integration frequencies of HDAdVs in primate ES cells were low (Ϸ2.7 ϫ 10 Ϫ5 /cell), the ratio of targeted to random integration using HDAdVs was higher compared with that of nonviral electroporation, which is consistent with our previous observation in mES cells (23). Although the ratio was 1:10 with HDAdV-mediated gene targeting even without negative selection, it was 1:50-600 for HPRT1 gene targeting by electroporation (11,12,17).…”

“…We reported previously that, by encoding long homologous sequences, HDAdV is an efficient and versatile gene targeting vector in mouse ES cells (23). To examine the efficiency of gene targeting by using HDAdVs in primate ES cells, we constructed a cynomolgus monkey HPRT1-targeting HDAdV plasmid, pBRHDAdrHPRTKO5-R, encoding a 13-kb region homologous to introns 3-8 of HPRT1, in which exons 5 and 6 were replaced with the IRES-␤geo-pA cassette ( Fig.…”

“…Because of the complete removal of viral genes from the vector genome, HDAdVs are generally less cytotoxic than E1-deleted AdVs, which allows them to be used at higher multiplicities of infection (MOIs) (22). In addition, we previously showed that the expanded cloning capacity of HDAdVs, which permits the insertion of larger segments of homologous DNA for HR, is advantageous in that it obtains highly efficient gene repair via HR in mES cells (23). The frequency of HR were extremely high at Ϸ2.2 ϫ 10 Ϫ3 per cell, and the percentage of HR to random integration was Ͼ50%.…”

Progress and prospects: gene transfer into embryonic stem cells

“…18 Other strategies to introduce transiently expressed genes could also be employed. Most of these alternative gene transfer techniques, including microinjection, adenovirus (Ad), 19 adeno-associated virus (AAV), herpesvirus or non-integrative retroviruses 20 have, however, not been assessed in hESCs, with the exception of Ad5 and AAV2. 21 Of note, the use of autonomously replicating extrachromosomal elements, such as plasmids containing an origin of replication, has recently been assessed in the context of hESCs.…”

“…37 Several strategies have been proposed to address the issue of low frequencies of gene targeting, aimed either at improving the transfection level, or at modifying the nature of the correcting DNA template. For instance, alternative transfection methods such as Ad-based 19 or microinjection have been used. Other methods, aimed at improving the overall rate of HR, have been assessed in somatic cells and should be assessed in the hESC setting, such as small fragment homologous replacement, 38 or triplex-forming oligonucleotides.…”