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Short interfering RNA (siRNA) therapeutics have soared in popularity due to their highly selective and potent targeting of faulty genes, providing a non‐palliative approach to address diseases. Despite their potential, effective transfection of siRNA into cells requires the assistance of an accompanying vector. Vectors constructed from non‐viral materials, while offering safer and non‐cytotoxic profiles, often grapple with lackluster loading and delivery efficiencies, necessitating substantial milligram quantities of expensive siRNA to confer the desired downstream effects. We detail the recombinant synthesis of a diverse series of coiled‐coil supercharged protein (CSP) biomaterials systematically designed to investigate the impact of two arginine point mutations (Q39R and N61R) and decahistidine tags on liposomal siRNA delivery. The most efficacious variant, N8, exhibits a twofold increase in its affinity to siRNA and achieves a twofold enhancement in transfection activity with minimal cytotoxicity in vitro. Subsequent analysis unveils the destabilizing effect of the Q39R and N61R supercharging mutations and the incorporation of C‐terminal decahistidine tags on α‐helical secondary structure. Cross‐correlational regression analyses reveal that the amount of helical character in these mutants is key in N8's enhanced siRNA complexation and downstream delivery efficiency. 

Abstract Successful clinical implementation of gene delivery relies on the use of viral or non‐viral based vectors to package and protect the therapeutic nucleic acid. These vehicles must also be able to direct the fate of the cargo once it has entered the cell to ensure that the nucleic acid is functional, and the desired outcome is achieved. Compared to viral vectors, non‐viral vectors have the advantage of incorporating different material types such as lipids, polymers, and peptides to tune overall safety and efficacy. Peptides are especially powerful when used in gene delivery vectors as they are able to increase gene delivery efficacy by introducing new biochemical functionality. This review will discuss the use of peptides as central design components in non‐viral gene delivery vectors. The contribution of the peptide component to the overall functionality of the delivery vehicle will be highlighted, with a focus on peptides as the only vehicle component or peptides in complex assemblies with lipids or polymers.