Overcoming the nuclear barrier in nonviral gene therapy: mechanisms, carrier design, and translational perspectives on NLS peptide and DNA targeting sequence strategies.

Efficient delivery of plasmid DNA (pDNA) into the nucleus remains a major bottleneck in nonviral gene therapy, particularly in post-mitotic and slowly dividing cells where the nuclear envelope constitutes a permanent barrier to passive entry. This review considers the two main strategies for active nuclear targeting: nuclear localisation signal (NLS) peptides, which recruit importin-α/β receptors directly to DNA cargo, and DNA nuclear targeting sequences (DTS), which exploit NLS-bearing transcription factors to mediate piggyback nuclear import. Rather than re-cataloguing nuclear import biology, we focus on the conditions under which each strategy succeeds or fails, incorporating advances in nuclear pore complex architecture and permeability, karyopherin-FG-nucleoporin electrostatics, and cytoplasmic constraints such as barrier-to-autointegration factor (BAF)-mediated clustering of exogenous DNA. NLS classes are compared with universal (SV40 enhancer), inducible (NF-κB) and tissue-specific (SMGA, SP-C, T1α) DTS elements, and four chemical coupling strategies are evaluated across peptide-, polymer- and lipid-based nanoassemblies. To address the near-absence of NLS/DTS mechanisms in current clinical pDNA programmes, we propose the Nuclear Accessibility Index (NAI): a three-axis framework integrating cell division status, carrier-DNA accessibility and intracellular signalling context. Treating the NAI as an axis vector rather than a summed score defines permissive, conditional and excluded regions of the design space. Two matter in practice: one in which highly proliferative cells render active targeting redundant, and one in which the rate-limiting problem is carrier engineering rather than the addition of a signal. The NAI offers design principles for nuclear-targeted nanocarriers and defines the preconditions for therapeutically relevant active import.