390. Phage c31 Integrase Induces Chromosomal Aberrations in Primary Human Fibroblasts
Authors: Jian Liu, Iben Jeppesen, Kate Nielsen, Thomas G. Jensen
Journal: Molecular Therapy (2006)

Phage c31 integrase is a recombinase with the potential to perform site-specific integration into a host chromosome. The enzyme mediates recombination between two different short sequences of DNA, attP and attB recognition sites. In mouse and human genomes, phage c31 integrase mediates the integration of plasmids bearing an attB site into sequences with partial sequence identity to attP, called pseudo attP sites. Based on the identification of phage c31 pseudo attP sites in the human genome it was estimated that the total number of human pseudo attP sites is between 102 and 103. Phage c31 integrase-mediated gene delivery is under investigation as an novel tool for human gene therapy. In order to investigate the safety of phage c31 integrase-mediated integration, we have studied potential effects of prolonged phage c31 integrase expression in primary human fibroblasts, derived from a spontanous abortion. Embryonic cells were used due to their high proliferative potential and genomic stability.Primary human fibroblasts were transfected with the plasmids pBabepuro or pBabepuroatt, containing the 285-base pair attB sequence, with or without the plasmid pCMVInt expressing the phage c31 integrase. The co-transfection experiments were performed with a 3:1 molar excess of pCMVInt compared to the pBabepuro/pBabepuroatt plasmids to increase the likelihood that pCMVInt plasmids were integrated in puromycin resistant cells. After puromycin selection, besides the cytogenetic analysis, DNA and RNA were also isolated and analysed using primers specific for integrase gene and also for the most common used integration site (the aatP site A on chromosome 8).Cells transfected with pBabepuro or pBabepuroatt alone had normal karyotypes. In contrast highly abnormal karyotypes were found in cells transfected with the pCMVInt plasmids. No obvious pattern involving specific chromosomes was observed, and chromosomes containing the previously published pseudo attP sites were not involved more frequently than the other chromosomes in the aberrations. Only 1 of 14 identified breakpoints matched the localisation of a published human pseudo attP site. Analysis of puromycin-selected clones expressing the integrase likewise showed abnormal karyotypes. The experiments were reproduced in primary human fibroblasts from another embryo, and again, abnormal karyotypes were exclusively found in cells transfected with plasmids containing the phage c31 integrase gene.Thus we have shown that the phage c31 integrase induces chromosome rearrangement in primary human cells raising serious safety concerns about possible clinical use. Another member of the family of site-specific recombinases, the Cre recombinase, has the ability to cause widespread DNA damage when expressed at high levels. Here pseudo-sites were suspected to act as target for the integrase and give rise to a mutagenic background. Since transgenic mice and Drosophila with phage c31 integrase expression have been generated with no adverse consequences, further work is required to understand the mechanisms behind phage c31 integrase mediated chromosome instability. Phage c31 integrase is a recombinase with the potential to perform site-specific integration into a host chromosome. The enzyme mediates recombination between two different short sequences of DNA, attP and attB recognition sites. In mouse and human genomes, phage c31 integrase mediates the integration of plasmids bearing an attB site into sequences with partial sequence identity to attP, called pseudo attP sites. Based on the identification of phage c31 pseudo attP sites in the human genome it was estimated that the total number of human pseudo attP sites is between 102 and 103. Phage c31 integrase-mediated gene delivery is under investigation as an novel tool for human gene therapy. In order to investigate the safety of phage c31 integrase-mediated integration, we have studied potential effects of prolonged phage c31 integrase expression in primary human fibroblasts, derived from a spontanous abortion. Embryonic cells were used due to their high proliferative potential and genomic stability. Primary human fibroblasts were transfected with the plasmids pBabepuro or pBabepuroatt, containing the 285-base pair attB sequence, with or without the plasmid pCMVInt expressing the phage c31 integrase. The co-transfection experiments were performed with a 3:1 molar excess of pCMVInt compared to the pBabepuro/pBabepuroatt plasmids to increase the likelihood that pCMVInt plasmids were integrated in puromycin resistant cells. After puromycin selection, besides the cytogenetic analysis, DNA and RNA were also isolated and analysed using primers specific for integrase gene and also for the most common used integration site (the aatP site A on chromosome 8). Cells transfected with pBabepuro or pBabepuroatt alone had normal karyotypes. In contrast highly abnormal karyotypes were found in cells transfected with the pCMVInt plasmids. No obvious pattern involving specific chromosomes was observed, and chromosomes containing the previously published pseudo attP sites were not involved more frequently than the other chromosomes in the aberrations. Only 1 of 14 identified breakpoints matched the localisation of a published human pseudo attP site. Analysis of puromycin-selected clones expressing the integrase likewise showed abnormal karyotypes. The experiments were reproduced in primary human fibroblasts from another embryo, and again, abnormal karyotypes were exclusively found in cells transfected with plasmids containing the phage c31 integrase gene. Thus we have shown that the phage c31 integrase induces chromosome rearrangement in primary human cells raising serious safety concerns about possible clinical use. Another member of the family of site-specific recombinases, the Cre recombinase, has the ability to cause widespread DNA damage when expressed at high levels. Here pseudo-sites were suspected to act as target for the integrase and give rise to a mutagenic background. Since transgenic mice and Drosophila with phage c31 integrase expression have been generated with no adverse consequences, further work is required to understand the mechanisms behind phage c31 integrase mediated chromosome instability.