PNMA2 is a human protein derived from the Ty3 retrotransposon that forms capsid-like nanocages and does not intrinsically package any nucleic acids, however it has previously been engineered to package RNA with low efficiency^[1].  In this project we apply protein engineering and directed evolution to repurpose PNMA2 for therapeutic mRNA packaging and delivery. We have engineered PNMA2 variants to package their own mRNA ‘genome’, constructed mutagenised plasmid libraries using error-prone PCR, expressed and purified capsid variants, and selected for RNase-resistant assemblies to enrich for RNA cargo protection and packaging efficiency, all according to a previously published method^[2][3]. Ongoing rounds of evolution will be guided by long-read sequencing and cryo-EM guided structural analysis, offering insight into both functional optimization and potential evolutionary trajectories of retroelement-derived proteins. Additionally, PNMA2 has an external 'spike' domain, which was shown to be immunogenic in mice^[4]. Through truncating the spike domain, we are evaluating it’s role in capsid assembly in order to engineer a less immunogenic capsid, also with the possibility to replace it and functionalise the surface of the capsid for enhanced delivery. In future work, we aim to evolve PNMA2 variants for improved cellular uptake, endosomal escape and cargo release. This approach not only expands the toolbox of RNA delivery technologies, but also provides a platform for exploring the evolution of virus-like capsids in a controlled, synthetic context.

[1] V. Madigan, Y. Zhang, R. Raghavan, M.E. Wilkinson, G. Faure, E. Puccio, M. Segel, et al., Proc. Natl. Acad. Sci. U.S.A., 2024, 121 (11) e2307812120

[2] N. Terasaka, Y. Azuma & D. Hilvert, Proc. Natl. Acad. Sci. U.S.A., 2018, 115 (21) 5432-5437

[3] S. Tetter, N. Terasaka, A. Steinauer, R. J. Bingham, S. Clark, A. J. P. Scott, N. Patel, et al., Science, 2021, 372, 6547, 1220–24. 

[4] J. Xu, S. Erlendsson, M. Singh, G. A. Holling, M. Regier, I. Ibiricu, J. Einstein, et al., Cell, 2024, 187, 4, 831-845.e19.