Sereno, Ferdinando; (2025) Engineering protein-based nanoparticles: Developing scalable expression and purification methods and mRNA-loading strategies for vaccine development. Doctoral thesis (Eng.D), UCL (University College London).

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Abstract

Encapsulins are prokaryotic nanocompartments that feature remarkable properties such as self-assembly, cargo loading capacity and resistance to harsh conditions, including high/low pH, high temperature and protease activity. The current state of the art explores numerous applications, such as vaccines and nanobioreactors, but the production methodologies are limited to the laboratory scale. The work presented in this thesis is divided in two areas, complementary with each other. The first aimed at repurposing encapsulins as mRNA carriers to develop a new class of thermoresistant mRNA vaccines. The second one consisted in the development of a complete bioprocess design, from upstream to downstream. This bioprocess standardised purification techniques, is scalable and addressed certain knowledge gaps related to encapsulin production, such as nonspecific nucleic acid loading. The design, applicable to the three main encapsulin architectures, outcompetes currently published work in terms of scalability, yield and purity and has been employed to produce and purify the mRNA-binding capsid variants. Loading of nucleic acid was achieved by fusion of a bacteriophage-derived, RNA-binding peptide to the interior of the encapsulin. A fluorescent RNA aptamer was encapsulated via an in vitro alkaline disassembly and reassembly procedure. Despite some shortcomings in specificity toward the cognate RNA sequence, this is the first study reporting in vitro loading of RNA inside an encapsulin. Protection of the cargo against nucleases and high temperature was confirmed. The recovery of loaded encapsulins was improved by reducing the RNA:capsid ratio. An alternative loading strategy, based on in vitro-triggered assembly, was shown to prevent non-specific nucleic acid loading but abolished aptamer fluorescence. This work presents the proof-of-concept and foundation for the future work, which will attempt the loading of a fully-fledged mRNA molecule test uptake and expression in antigen-presenting cells.

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