Progress and prospects: nuclear import of nonviral vectors

Abstract

The nuclear envelope represents a key barrier to successful nonviral transfection and gene therapy both in vitro and in vivo. Although the main purpose of the nuclear envelope is to partition the cell to maintain cytoplasmic components in the cytoplasm and nuclear components, most notably genomic DNA, in the nucleus, this function poses a problem for transfections in which exogenous DNA is delivered into the cytoplasm. After delivery to the cytoplasm, nucleic acids rapidly become complexed with cellular proteins that mediate interactions with the cellular machinery for trafficking. Thus, it is these proteins that, in essence, control the nuclear import of DNA, and we must also understand their activities in cells. In this review, we will discuss the principles of nuclear import of proteins and DNA-protein complexes, as well as the various approaches that investigators have used to improve nuclear targeting of plasmids. These approaches include complexation of plasmids with peptides, native and engineered proteins, ligands and polymers, as well as the inclusion of transcription factor-binding sites for general and cell-specific delivery.

Keywords: nonviral gene transfermid R:plasmidmid R:nuclear pore complexmid R:importinmid R:nuclear localization signalmid R:karyopherin.

Figure 1

Mechanisms of protein nuclear import. Cargos targeted for nuclear import contain NLSs on their surface which interact with a number of distinct importin-α/β heterodimers or importin-β isoforms directly. These complexes are targeted to and translocated across the nuclear envelope through the NPC. Upon reaching the nucleus, RanGTP binds to importin-β, causing a conformational change that releases the bound cargo. The importins are then recycled to the cytoplasm as a RanGTP–Importin-β complex or, in the case of importin-α, by its export carrier CAS. The complex then dissociates in the cytoplasm after the hydrolysis of RanGTP, which is facilitated by RanGAP at the cytoplasmic face of the NPC. RanGDP is then imported to the nucleus by NTF2 and the guanine exchange factor RCC1 converts it to RanGTP.

Figure 2

Protein-mediated plasmid nuclear import. Transcription factors and other nuclear proteins normally enter the nucleus through interactions between their NLSs and importin family members. However, if plasmids containing certain sequences that act as scaffolds for transcription factors and other DNA binding proteins (termed ‘DTS’, or DNA nuclear targeting sequences) are deposited into the cytoplasm during transfection, they can form complexes with these proteins, thereby attaching NLSs to the DNA. Some, but not all, of these NLSs may be in a conformation able to interact with importins for transport of the DNA–protein complex into the nucleus through the nuclear pore complex.

Figure 3

Cell-specific plasmid nuclear import. Certain DNA nuclear targeting sequences have been shown to act in cell-restricted manners. In the case of the smooth muscle γ-actin promoter, which acts as a smooth muscle cell DTS, it has been shown that two key factors that are coexpressed in smooth muscle, SRF and Nkx3, form complexes with the plasmid leading to an importin-recognizable complex that can be localized to the nucleus. By contrast, in nonsmooth muscle cells that do not express one or the other of these factors, an importin-binding complex is not formed leading to greatly reduced nuclear import.

Figure 4

Methods to enhance plasmid nuclear import. A number of different approaches have been developed to promote recognition of plasmids by importin family members to increase nuclear import. These include peptide-nucleic acid clamp-conjugated NLS peptides bound to DNA, sequence-specific DNA binding proteins bound to DNA, NLS peptides covalently attached to DNA and NLS peptides electrostatically bound to DNA.