Oral Presentation The 16th Australian Peptide Conference 2025

Engineering a Tissue-Targeted Delivery Platform for DNA, mRNA, siRNA, and CRISPR/Cas9: Optimising In Vivo Efficacy and Hard-to-Transfect Cell Uptake (129414)

Peter M Moyle 1 , Hong Yi Lau 1 , Limin Dai 1 , Frazer Sinclair 1 , Anjuman A Begum 1 , Istvan Toth 2
  1. School of Pharmacy and Pharmaceutical Sciences (SPPS), The University of Queensland, Woolloongabba, QLD, Australia
  2. School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD, Australia

Gene delivery is a transformative strategy for preventing, treating, and potentially curing diseases that are inaccessible to conventional pharmaceutical interventions. It also plays a crucial role in elucidating biological mechanisms and accelerating drug development. Peptide-based gene delivery platforms offer a versatile alternative to lipid nanoparticles (LNPs), enabling distinct cellular uptake mechanisms and customizable targeting features. However, challenges such as in vivo instability and limited tissue specificity continue to hinder clinical translation.

Our laboratory has developed modular, multicomponent peptide-based delivery systems for a range of nucleic acid payloads (DNA, mRNA, siRNA, and CRISPR/Cas9). These systems incorporate:

  1. Cationic peptides and/or polymers to condense oligonucleotides,
  2. Endosomal escape motifs to promote cytosolic release, and
  3. Targeting ligands selected for binding and uptake via tumour-associated receptors.

All components are efficiently synthesised using automated Fmoc solid-phase peptide synthesis. When combined with selected phospholipids, these systems form nanoparticles that exhibit synergistic enhancements in targeted cellular uptake, robust endosomal escape, and high transfection efficiency (comparable to Lipofectamine 3000) under both serum-free and high-serum conditions.

Systematic evaluation of phospholipid combinations (cationic, anionic, and zwitterionic) enabled the identification of formulations optimised for high serum environments, such as in vivo conditions. Further refinement involved tuning the component ratios to modulate nanoparticle size, uptake, and gene expression, along with the introduction of polymer coatings and freeze drying to improve serum stability and protect both carrier and cargo from degradation.

Transfection studies using plasmid DNA and mRNA encoding fluorescent reporters demonstrated potent gene expression in cancer cell lines, even under increasing serum concentrations. Importantly, receptor-targeted formulations selectively transfected cell lines overexpressing the corresponding receptor in both in vitro and in vivo experiments. In hard-to-transfect cell lines, these peptide-based systems outperformed Lipofectamine 3000 in both uptake and gene expression efficiency.

These results highlight the promise of our delivery platform for targeted, in vivo gene therapy applications, and as a powerful tool for research involving difficult-to-transfect cell types.