Abstract
Nonviral DNA delivery has emerged as a promising alternative to viral vectors for gene therapy because of its reduced immunogenicity, scalable manufacturing, and capability of loading larger cargos. Despite these advantages, multiple hurdles must be addressed. The endosomal barrier poses a significant obstacle because DNA is often degraded in acidic vesicles following endocytosis. After endosomal release, DNA is sensed by the cyclic GMP-AMP synthase, a cytosolic immune sensor, leading to inflammation and lower gene expression levels. Furthermore, DNA requires nuclear entry for gene expression, which has been one of the most complex challenges for the field to progress. Recent advances have explored innovative strategies to overcome these hurdles for nonviral DNA delivery. This review highlights the current landscape of nonviral DNA delivery, focusing on intracellular barriers and recent technological breakthroughs. We further discuss future directions to overcome these challenges to develop safe, effective, and clinically translatable nonviral DNA delivery systems for a range of therapeutic applications. SIGNIFICANCE STATEMENT: Nonviral DNA delivery offers a safer, scalable alternative to viral vectors but faces key challenges such as endosomal escape, poor tolerability, and low levels of transgene expression. This review highlights the critical barriers and emerging solutions, outlining how recent advances can enable low-cost, redosable genetic medicine and DNA vaccines suited for clinical translation.