Towards the development of next-generation MergoTM — a nucleic acid-based delivery platform

Abstract

Nucleic acids offer great potential for therapeutic and drug delivery applications, but their clinical translation is hindered by several biological barriers. This thesis addresses some of these challenges by optimising Sixfold's Mergo system, a self-assembled RNA construct, for the in vivo delivery of siRNAs. Focusing on stability, cellular uptake, endosomal escape, and off-target effects, we performed a comprehensive investigation of Mergo's performance in different biological matrices.To ensure efficient delivery of intact therapeutics, stabilising Mergo systems against nucleolytic degradation is essential. Through detailed analysis of Mergo and other RNA constructs in biological matrices, we identified vulnerable regions and implemented strategies to enhance nuclease resistance. These strategies included specific chemical modifications and the design of novel linkers engineered for controlled siRNA release at the target site.Further investigation focused on the critical steps of cellular uptake and endosomal escape, which are known bottlenecks for nucleic acid-based therapies. While cellular uptake of Mergo was readily achieved, endosomal escape was identified as the rate-limiting steps for Mergo in vitro.Finally, to assess the safety and efficacy of Mergo, we employed a combination of in silico, in vitro, and in vivo studies to evaluate both on- and off-target effects. This work not only provided preliminary insights into Mergo's potential for therapeutic delivery but also established a valuable methodology for evaluating future RNA-based delivery systems and therapeutics.In summary, this thesis investigates the challenges and opportunities of the Mergo delivery system. By addressing key barriers, this work aims to accelerate the development of Mergo as a promising delivery platform for clinical applications. Furthermore, as Mergo is RNA-based, this research contributes to a broader understanding of the challenges and opportunities within the RNA delivery and therapeutics field