289. Sequence Modified Antibiotic Resistance Genes Provide Sustained Plasmid Mediated Transgene Expression in Mammals

Abstract

Conventional plasmid vectors are incapable of achieving sustained levels of transgene expression in vivo. Even in quiescent tissues where plasmid DNA levels remain static, transgene expression is transcriptionally extinguished or silenced over a period of several weeks. Extensive work has led to the finding that the length (~1kb or more) rather than the sequence of DNA contained outside of the eukaryotic expression cassette (between 5’end of the promoter and 3’end of the poly A site- such as a canonical bacterial plasmid backbone) is the critical factor that dictates transcriptional silencing in vivo (Lu, J et al. 2012). Nucleosomal patterns are considered a key element in the establishment and maintenance of silenced chromatin. An/Tn sequence motifs are known to substantially enhance nucleosome mobility and enhance RNA polymerase II accessibility. These sequences sometimes referred to as nucleosome exclusion, remain a mystery in terms of their mode of action. Therefore, we explored whether the addition of a higher number of An/Tn sequences would influence plasmid-mediated transcriptional silencing. First, we found that the placement of a stretch of 20 ‘T’ nucleotides into every 60bp of a random 2.2kb fragment of DNA used in place of the bacterial plasmid backbone abrogated transcriptional silencing. Next, we optimized the An/Tn sequence content of the plasmid containing Kan and Amp bacterial antibiotic resistance markers without altering their protein coding sequence. These modified antibiotic resistance markers did not affect their propagation in bacterial culture. Most importantly, when transfected into mouse liver they were capable of producing sustained transgene expression at levels 10 times higher than a conventional plasmid. These plasmids maintain expression at levels we previously obtained with minicircle vectors (DNA vectors lacking a bacterial plasmid backbone) without the requirement for special cell lines or plasmid modifications. Thus, we have created a generic plasmid vector that can be produced using standard production schemes that provide sustained expression in quiescent cells and tissues that will be useful in future gene transfer applications.