Self-replicating RNAs, termed replicons, are a promising new mode for vaccines. Replicons amplify within host cells and enable sustained antigen expression from a single injection. Previous studies have shown that prolonged antigen availability can positively impact multiple facets of the immune responses to vaccination. Therefore, replicon-based vaccines provide a practical approach to extending vaccine exposure by regulating antigen and adjuvant kinetics. This project aims to develop gene circuits activated by trimethoprim (TMP), an FDA-approved small molecule drug, to regulate the expression of vaccine immunogens from replicons. Gene expression was regulated through the addition of a destabilization domain (DDd) from the bacterial dihydrofolate reductase to the immunogen-encoding gene of interest or non-structural proteins (nsPs) that form the replicon’s amplification machinery. DDd is intrinsically unstable and is targeted – along with the fused immunogen or nsPs – for degradation by proteasomes unless it is stabilized by TMP. Using firefly luciferase (fLuc) as a reporter cargo, we designed and evaluated different TMP-responsive circuits with DDd fused to one of the four nsPs of the replicon backbone alone or in combination with the direct fusion of DDd to fLuc. For in vivo administration, replicons are encapsulated into lipid nanoparticles using a microfluidic device. Among different circuits tested, a gene circuit with DDd fused to both nsP3 and fLuc (nsP3-DD + DD-fLuc circuit) offered the strongest regulation in transgene (fLuc) expression as indicated by the bioluminescence signal, resulting in ~60-fold difference in bioluminescence between ‘on’ (+TMP) and ‘off’ (-TMP) states in vitro in C2C12 mouse skeletal muscle cells. Following intramuscular injection in mice, the nsP3-DD + DD-fLuc circuit showed proportional modulations in bioluminescence in response to the TMP dose (provided in the diet) with up to two orders of magnitude difference in bioluminescence between no and high TMP regimens. In addition, the response to TMP was reversible such that the fLuc level remained low until TMP was added and returned to low levels when TMP was removed. We used this replicon gene circuit for the expression of a clinical HIV vaccine candidate immunogen, the engineered outer domain (eOD) of HIV envelope gp120 protein. We demonstrated that the output antibody responses to immunization are modulated in responses to the TMP dose. We found that the gene circuit with DDd fused to both nsP3 and fLuc (nsP3-DD + DD-fLuc circuit) offers significant regulation of gene expression, allowing for immunization responses to be modulated by TMP dose through the regulated expression of clinical HIV vaccine candidate immunogen eOD.
