372. Targeting of Non-Viral Vectors to Specific Subnuclear Domains

Abstract

Top of pageAbstract The cell biology principles and theories that define mechanisms behind eukaryotic transcription can potentially be applied to non-viral gene therapy. With this approach, we have demonstrated that non-viral vectors can be engineered to traffic to specific subnuclear domains. Non-viral vectors are mainly designed for three things, to efficiently deliver a gene to a target tissue or cell, to mediate plasmid nuclear import, and to ultimately express a transgene. Therefore, the focus of non-viral gene therapy research often lay with global delivery methods and successful translocation of the plasmid into the nucleus. As a result, the study of intranuclear plasmid behavior is often neglected, despite the fact that the nucleus is a highly ordered and organized compartment. Gene expression of endogenous chromatin is partially controlled by the segregation of euchromatin from heterochromatin and the maintenance of distinct, subnuclear chromosome territories. Development of non-viral vectors that effectively target to regions of the nucleus where transcription is already maximized could enhance and increase the overall efficacy of gene therapy. To test this hypothesis we microinjected nuclei with plasmids that contained different DNA regulatory sequences. Microinjected cells were then fixed at different time points so fluorescence in situ hybridizations and immunocytochemistry could be performed. Based on qualitative and quantitative 3-D analysis of confocal and deconvolved images, we found that the addition of different regulatory elements changed the intranuclear trafficking patterns of plasmids. Plasmids containing RNA polymerase I promoters localized into large speckles within the nucleolus and strongly co-localized with fibrillarin and upstream binding factor (UBF). Plasmids with RNA polymerase II promoters trafficked to nucleoplasmic regions rich in mRNA processing machinery. In contrast, plasmids without eukaryotic regulatory sequences remained diffuse throughout the nucleoplasm and did not co-localize with any of the marker proteins used. Further manipulation of our non-viral vectors altered plasmid intranuclear trafficking patterns and localization. Transgene expression could potentially be enhanced and prolonged if we can target non-viral vectors into specific subnuclear domains beyond the nuclear envelope and sustain their co-localization with active transcriptional machinery.