In trans RNA replication of a defective self-amplifying RNA lowers RNA replication but enhances protein translation.

RNA replication is fundamental to the life cycle of the large majority of viruses and has attracted increasing interest due to its potential to enhance mRNA expression for vaccines and gene therapy. This process relies on RNA-dependent RNA polymerase (RdRp) genes that, once translated, recognize their cognate mRNAs and replicate them. Self-amplifying RNAs (saRNAs) - "replicons" - can be engineered by incorporating the RdRp gene, a gene of interest (GOI), and the required cis-acting sequence elements for replication. Compared to non-replicating RNAs, saRNAs typically achieve up to two orders of magnitude higher gene expression in target cells. However, this enhanced replication is often associated with high cytotoxicity. Developing strategies to control or modulate RNA replication is therefore critical for the therapeutic application of saRNAs. Here, we report the effects of combining replication-competent and replication-deficient replicons on RNA replication and GOI translation. We quantify the extent to which the competent replicon rescues replication of the deficient replicon in trans, leading to reduced overall RNA replication but to increased total GOI translation. These findings highlight the delicate balance between RNA replication and protein synthesis in the context of cellular cytotoxity and metabolic burden, factors to be considered when designing strategies of saRNA-based therapeutics.