Improving the dynamic range of a trimethoprim-responsive self-amplifying RNA

Self-amplifying mRNA (SAM) is a potential platform for protein replacement, as it enables recipients to transiently produce a therapeutic protein for 4-8 weeks. To obtain external control over the level and duration of protein expression, synthetic SAM constructs that switch OFF in the presence of a small molecule such as trimethoprim (TMP) have been developed in the past. Here, we increase the ON-state protein expression of such a TMP-responsive SAM by fusing the effector protein L7Ae to a TMP-responsive destabilizing domain (DD) on both terminals. We also lower the OFF-state by adding a duplicate of the SAM 3'UTR downstream of the DD-L7Ae subgenomic ORF. We demonstrate that fusing two DDs to L7Ae has a beneficial effect on the response rate and level of protein production after removal or addition of TMP. We further demonstrate that overexpression of DD-L7Ae results in microscopic abnormalities. These insights are implemented in a mechanistic model, and further improvements of the platform are highlighted.