Gene therapy, while offering a unique means to silence disease associated genes and repair genetic defects, is currently hindered by the inefficient delivery and trafficking of oligonucleotide therapeutics (e.g. siRNA or plasmid DNA (pDNA)) to their required intracellular site of action. Barriers to efficient delivery of oligonucleotide therapeutics include their charge; hydrophilicity; size; metabolic and immune-mediated clearance; need for cellular uptake (without endo(lyso)somal entrapment); and the requirement for pDNA translocation into the nucleus. To overcome these barriers, we have investigated a multi-component approach, including: i) cationic poly-L-lysine dendrons (up to +32 charge), for oligonucleotide condensation, improved cell uptake and metabolic stability; ii) cell penetrating peptides (CPPs; TAT(48-60), NRTN, SPACE) or receptor-targeted peptides (bombesin) to improve intracellular delivery; iii) lipidation, to improve stability, promote nanoparticle formation, and improve cellular uptake; iv) nuclear targeting species, to improve DNA delivery into the nucleus for transcription; and iv) endosome release agents (GALA, KALA, HA2, poly-histidine), to increase oligonucleotide delivery into the cytoplasm.
Libraries of defined, highly pure multicomponent delivery systems have been produced using orthogonal maleimide and copper(I)-catalysed alkyne-azide cycloaddition (CuAAC) chemistries, enabling the simple variation of components attached to a dendron-CPP backbone. Using these libraries we have assessed the effect of each component on cellular uptake, endosome escape, intracellular localisation, oligonucleotide stability, cellular toxicity, particle size and charge, as well as reporter gene transcription or knockdown with pDNA or siRNA respectively. The information obtained from these experiments has provided information on which component mixtures are most suited to the delivery of pDNA or siRNA, with these mixtures forming the basis for further optimisation.