1103. Site-Specific Chromosomal Integration in Human Mesenchymal Stem Cells Mediated by PhiC31 Integrase

Abstract

Human mesenchymal stem cells (hMSCs) have emerged as a promising new tool in gene therapy/ cell therapy strategies due to their potential use in a wide variety of applications, including both short- and long-term regeneration. With viral vectors being frequently criticized for their immunogenicity, random integration and difficult production, alternatives are desirable. A safe and effective gene delivery method resulting in targeted chromosomal integration with prolonged therapeutic levels of gene expression would be valuable. PhiC31 integrase, a site-specific recombinase from phage phiC31, pairs an attB recognition site on an incoming plasmid with pseudo attP sites in mammalian genomes. This integrase has been used to achieve genomic integration in human keratinocytes, mouse liver, rat retina and rabbit joints. Here we describe non-viral approaches for gene delivery using phiC31 integrase, transfected either as mRNA or as a DNA expression vector, in conjugation with an attB-containing donor plasmid. We evaluated the efficacy of mRNA encoding phiC31 integrase to mediate unidirectional integration into pseudo attP sites when co-transfected with an attB donor plasmid containing a transgene of interest. We also demonstrated that co-transfection of a plasmid expressing phiC31 integrase with a plasmid containing the luciferase marker gene and an attB site resulted in 39-fold higher expression of luciferase as assessed by live imaging, compared to control transfections without integrase. Expansion of the integrants and their differentiation into the osteogenic phenotype revealed sustained gene expression, indicating that the integration sites used in undifferentiated hMSCs continued to provide gene expression after differentiation of the cells. Finally, plasmid rescue and DNA sequence analysis of the integration sites showed that the integration events occurred at several pseudo attP sites in the human genome. Summarizing our results, phiC31 integrase, provided either as mRNA or DNA, mediates site-specific integration of transgenes and can provide high levels of gene expression in hMSCs and differentiated osteogenic cells. These findings suggest that phiC31 integrase represents an efficient and practical non-viral approach for genetic modification of hMSCs that could have potential for clinical applications.