UC San Diego

This dissertation describes the development and application of DNA-peptide conjugates to improve non-viral gene delivery by addressing intracellular barriers that limit DNA therapeutics. Chapter 1 introduces the promise of DNA-based gene therapy and frames nuclear localization as a major bottleneck in non-viral DNA delivery. Chapter 2 describes the development of DNA-PepTAG, a chemoenzymatic platform for generating site-specific peptide-modified DNA gene cassettes. Chapter 3 demonstrates that nuclear localization signal-modified gene cassettes enhance reporter and therapeutic protein expression in growth-arrested mammalian cells. Chapter 4 investigates the mechanistic basis of this enhancement, showing that NLS-modified gene cassettes improve nuclear DNA accumulation and gene expression through importin-dependent trafficking. Chapter 5 expands the DNA-PepTAG platform toward additional applications, including HDR templates for genome editing, endosomal escape peptide-modified gene cassettes, and self-transcribing DNA constructs. Chapter 6 summarizes the broader implications of engineering DNA cargos as active participants in their own delivery and highlights future opportunities for non-viral gene therapy.