Progress and prospects: nuclear import of nonviral vectors

Purpose: The nuclear envelope remains a major barrier to successful nonviral transfection and gene therapy. After exogenous DNA is delivered to the cytoplasm, it rapidly complexes with cellular proteins that control its intracellular trafficking and nuclear import. Understanding these mechanisms is necessary to improve plasmid nuclear targeting and gene expression.

Hypothesis: No formal experimental hypothesis. Central thesis: nuclear import of plasmid DNA is a key rate-limiting barrier for nonviral gene delivery; plasmids can enter the nucleus through the nuclear pore complex in a sequence-specific manner via DNA nuclear targeting sequences (DTSs) that bind NLS-containing transcription factors, and exploiting these mechanisms—or using NLS peptides, nuclear proteins, ligands, polymers, or NPC modulation—can enhance nuclear delivery and expression.

Aims: Discuss the principles of nuclear import of proteins and DNA–protein complexes. - Review approaches used to improve nuclear targeting of plasmids. - Summarize methods including peptide complexation, native and engineered proteins, ligands and polymers, and transcription factor-binding sites for general and cell-specific delivery. - Highlight progress and prospects in proteomics, RNA interference, in vivo imaging, and designer proteins for nonviral nuclear import.

Delivery system: Payload: plasmid DNA (supercoiled), linear DNA, fluorescently labeled plasmids, DNA–protein complexes. - Nonviral vectors/carriers: naked DNA, lipoplexes, polyplexes, polyethyleneimine (PEI), PEGylated polylysine/lipid/DNA nanoparticles, PAMAM dendrimers, Pluronics, calcium phosphate transfection. - Nuclear import enhancers: NLS peptides (classical SV40 large T-antigen NLS, bipartite NLS, noncanonical), peptide nucleic acid (PNA) clamps, DNA-binding proteins (NF-κB p50, SRF, Nkx3.2, histone H2B, NM23-H2, Chx10), dexamethasone–glucocorticoid receptor conjugates, TCHD (trans-cyclohexane-1,2-diol). - DTS elements: SV40 72-bp enhancer repeat, NF-κB binding sites, smooth muscle γ-actin (SMGA) promoter, cell-specific promoters for osteoblasts, endothelial cells, alveolar type II epithelial cells, smooth muscle cells. - Target cells/tissues: cultured fibroblasts, HeLa, HEK-293, HepG2, U373, smooth muscle cells, endothelial cells, airway/alveolar epithelial cells, oligodendrocytes, neuroblastoma SYSY, HuH-7, 16HBEo; in vivo vasculature, skeletal muscle, lungs.

Approach: Review of preclinical literature; no primary experiments. Discusses microinjection into cytoplasm vs nucleus, transfection with lipoplexes/polyplexes, digitonin-permeabilized cell assays, proteomic affinity chromatography, FRET, RNA interference knockdown, and in vivo plasmid delivery to animal tissues. Models include dividing and nondividing cultured cells and living animals; no clinical trials.

Key methods: Microinjection followed by reporter gene expression. - Quantitative PCR, Southern blot, electron microscopy for nuclear plasmid quantification. - Fluorescence microscopy, FRET for nuclear import and complex behavior. - Proteomics: plasmid affinity chromatography, 2D SDS-PAGE, LC-MS/MS. - RNA interference knockdown of importins and transcription factors; Western blot. - Whole-animal luminescence/fluorescence imaging. - Nuclear pore complex modulation with TCHD.

Key results: Nuclear microinjection of plasmids yielded expression in 50–100% of mouse fibroblasts; cytoplasmic injection largely failed. - 30–100 times more DNA delivered to cytoplasm was needed for equivalent expression versus nuclear delivery, even in dividing cells. - Only 1–10% of unmodified plasmids delivered to cells were detected in the nuclear fraction. - SV40 72-bp DTS mediated plasmid nuclear import in all cells tested and enhanced gene expression in vasculature, skeletal muscle, and lungs in vivo. - NF-κB binding sites increased expression 12-fold; FRET showed ~60 times more plasmids entered the nucleus when multiple NF-κB sites were present. - SMGA DTS (176 bp) drove smooth muscle–specific nuclear import via SRF and Nkx3.1/3.2; mutation or RNAi abolished import. - Proteomics with SMGA DTS identified 274 unique proteins vs 41 with control plasmid; importin β was required for nuclear import, while importin 7 knockdown had little effect. - Histone H2B and NM23-H2 stimulated DNA nuclear import over 6-fold in reconstituted assays. - Dexamethasone conjugation to PAMAM or PEI increased expression 20–40-fold, correlated with nuclear localization. - PEGylated polylysine/lipid/DNA nanoparticles entered nuclei within 15 min; import decreased when minor diameter exceeded 25 nm. - TCHD increased nuclear localization of plasmids but caused 50% viability decrease in Vero cells at 3% w/vol.

Interpretation: The nuclear envelope is a major barrier to nonviral gene delivery. Sequence-specific DTSs can recruit endogenous NLS-containing transcription factors to mediate plasmid nuclear import, enabling general or cell-specific targeting. Proteomics, RNAi, and in vivo imaging are expected to identify key transport proteins and mechanisms. The authors argue that understanding DNA–protein complexes will allow rational design of vectors and designer proteins with DNA-binding domains and spatially distinct NLSs. However, whether nuclear import is rate-limiting in vivo remains unresolved.

Limitations: Review article; no primary data. - Many studies use microinjection or cultured cells; relevance to in vivo systemic delivery is uncertain. - NLS peptide approaches show inconsistent results; peptide–DNA complexes may bury NLSs or reduce transcription. - Covalent DNA labeling can decrease transcriptional activity and alter trafficking. - TCHD modulation of NPC gating may cause toxicity and affect all cellular protein trafficking. - Immunogenicity risk of peptides in vivo may limit repeat administration. - Most work empirical rather than mechanistic; no clinical trials or large-animal validation discussed. - Whether nuclear import is a rate-limiting barrier in vivo remains unresolved.