Optimization of Streptomyces bacteriophage φC31 integrase system to prevent post integrative gene silencing in pulmonary type II cells

Abstract

AbstractφC31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study aimed at optimizing elements of φC31 integrase system for alveolar type II cells. Luciferase and β-galactosidase activities were measured at different time points post transfection. 5-Aza-2'deoxycytidine (AZA) and trichostatin A (TSA) were used to inhibit DNA methyltransferase and histone deacetylase complex (HDAC) respectively. In A549 cells, expression of the integrase using a CMV promoter resulted in highest integrase activity, whereas in MLE12 cells, both CAG and CMV promoter were equally effective. Effect of polyA site was observed only in A549 cells, where replacement of SV40 polyA by bovine growth hormone (BGH) polyA site resulted in an enhancement of integrase activity. Addition of a C-terminal SV40 nuclear localization signal (NLS) did not result in any significant increase in integrase activity. Long-term expression studies with AZA and TSA, provided evidence for post-integrative gene silencing. In MLE12 cells, both DNA methylases and HDACs played a significant role in silencing, whereas in A549 cells, it could be attributed majorly to HDAC activity. Donor plasmids comprising cellular promoters ubiquitin B (UBB), ubiquitin C (UCC) and elongation factor 1α (EF1α) in an improved backbone prevented post-integrative gene silencing. In contrast to A549 and MLE12 cells, no silencing could be observed in human bronchial epithelial cells, BEAS-2B. Donor plasmid coding for murine erythropoietin under the EF1α promoter when combined with φC31 integrase resulted in higher long-term erythropoietin expression and subsequently higher hematocrit levels in mice after intravenous delivery to the lungs. These results provide evidence for cell specific post integrative gene silencing with φC31 integrase and demonstrate the pivotal role of donor plasmid in long-term expression attained with this system.

Blastocyst Formation Rate and Transgene Expression are Associated with Gene Insertion into Safe and Non-Safe Harbors in the Cattle Genome

“…Promoter methylation is a powerful cause for transgene silencing in vitro and in vivo due to methylation of CpG dinucleotide by DNA methyltransferase enzymes. One solution to this problem is the removal of the CpG motifs in the promoter and bacterial backbone, which leads to stable, uniform expression of integrative gene in the genome of target cells 27 . Using CpG-free EF1α promoter, we observed that EGFP expression level was higher than that of wild-type EF1α and CMV promoters from BF10 site in blastocysts (Fig.…”

α-l-iduronidase gene-based therapy using the phiC31 system to treat mucopolysaccharidose type I mice

“…Based on this observation, we constructed several vectors with CMV‐derived promoters (Figure ) and monitored their expression for more than 1 month to validate them for long‐term gene expression in vitro . Although all integrative vectors maintained gene expression for more than 1 month, we decided to use pattB‐CAG‐IDUA in all of our in vivo experiments because the hybrid CMV promoter CAG has been used in several in vivo gene transfer experiments without observing gene silencing , whereas the pure CMV promoter is typically silenced . Additionally, Aronovich et al .…”

Site-Specific Recombination Using PhiC31 Integrase

“…However, this increased recombination activity has not been replicated. Rather, two recent studies have concluded that adding an NLS to the C-terminal end of f C31 integrase does not signi fi cantly increase activity, and instead causes a decrease in activity (Aneja et al 2009 ;Woodard et al 2010a ) .…”

“…Using cellular promoters, however, resulted in stable, long-term expression of the integrated transgene (Aneja et al 2009 ) . Thus, whereas it may not be useful to optimize the coding sequence of f C31 integrase or to add a NLS sequence to either end of the protein, it may be advantageous to optimize the donor plasmid by using a cellular promoter for transgene expression.…”

“…A concern that led to this modi fi cation was the possibility that the presence of CpG nucleotides and bacterial sequences in the donor plasmid backbone could lead to post-integrative silencing of the transgene. A recent study attempted to address post-integrative gene silencing by reducing the number of CpG dinucleotides in the plasmid backbone and by using cellular promoters to drive transgene expression, rather than the viral cytomegalovirus promoter (Aneja et al 2009 ) .…”