Gene silencing therapies have shown success in suppressing the translation of pathogenic target proteins and, hence, this strategy holds great promise for the treatment of numerous central nervous system (CNS) disorders. Endeavours arising from our current knowledge of multimolecular delivery vehicles focus on overcoming the difficult delivery of siRNA to target tissues due to issues relating to anatomical accessibility, slow diffusion, poor safety, and the requirement for precise cell uptake within the unique CNS environment. To address this challenge, the present work proposes the use of novel poly-L-ornithine (PLO) derivatives to construct polyplexes as non-viral vectors. As a proof-of-concept therapeutic approach, a putative regenerative therapy for multiple sclerosis was pursued. Death receptor 6 (DR6) is described as a negative regulator of oligodendrocyte precursor cell (OPC) survival, maturation and myelination, and its attenuation has been shown to boost remyelination in vivo.<br/>We obtained well-defined PLO through a robust and scalable polymerization procedure. We found that PEG derivatization failed to alter the second structure of the main chain and observed derivative degradation by proteases. All derivatives generated formed complexes with siRNA(30-60nm) and slightly positive Z-potential, with said polyplexes displaying stability under in vitro/in vivo scenarios. The physicochemical and biological properties of our novel nanocarriers are exhaustively characterized, highlighting their unique biodegradability and biocompatibility properties and effective gene silencing in primary OPCs. Our polyplexes efficiently silenced DR6 at the gene and protein level (Fig. 1), with an associated downregulation in cell death. However, protein and immunocytochemistry analysis showed that this intervention did not translate into OPC maturation. The Px2 (PLO-PEG3000) polyplex exhibited the highest efficiency, leading to a reduction in DR6 and PARP protein expression.

Polypeptide-based polyplexes to boost effective gene silencing in CNS disorders

2019 Fall National Meeting

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