Nuclear entry of nonviral vectors

Abstract

Nonviral gene delivery is limited to a large extent by multiple extracellular and intracellular barriers. One of the major barriers, especially in nondividing cells, is the nuclear envelope. Once in the cytoplasm, plasmids must make their way into the nucleus in order to be expressed. Numerous studies have demonstrated that transfections work best in dividing populations of cells in which the nuclear envelope disassembles during mitosis, thus largely eliminating the barrier. However, since many of the cells that are targets for gene therapy do not actively undergo cell division during the gene transfer process, the mechanisms of nuclear transport of plasmids in nondividing cells are of critical importance. In this review, we summarize recent studies designed to elucidate the mechanisms of plasmid nuclear import in nondividing cells and discuss approaches to either exploit or circumvent these processes to increase the efficiency of gene transfer and therapy.

Figure 1

Plasmid nuclear import in nondividing cells. Plasmids were labeled using a Cy5-PNA clamp and microinjected into the cytoplasm of TC7 cells as previously described.^, The DNA can be seen to begin to enter and accumulate in the nucleus as early as 40 min following cytoplasmic injection. If the cells are allowed to incubate for longer times, up to 100% of the PNA-labeled DNA is detected within the nucleus. Arrows indicate nuclei in two injected cells.

Figure 2

Model for sequence-specific DNA nuclear import. Once in the cytoplasm, plasmids containing a DTS (depicted in yellow) can interact with newly synthesized transcription factors to form protein–DNA complexes. Since transcription factors contain NLSs for their nuclear localization, the DTS-containing DNA can become coated at distinct sites with one or more NLS, which can then interact with the importins to mediate nuclear import of the entire complex. By contrast, plasmids lacking a DTS cannot form the appropriate DNA–protein complexes for nuclear import.

Figure 3

Various approaches to increase nuclear localization of transfected plasmids. A number of methods have been proposed and tested to increase the nuclear import of plasmids following transfection. Most rely on the addition of NLS-peptides or NLS-containing proteins to the DNA, by either electrostatic, covalent, or PNA clamps, to increase nuclear import. Other approaches have used general or cell-specific DTSs (in yellow), or synthetic DTS/NLS-protein pairs (in blue).