Temporally-controlled gene expression via small molecule-regulated self-replicating RNA for cancer immunotherapy

Introduction:: Self-replicating RNAs, termed replicons, are emerging as an attractive RNA modality for gene delivery and cancer immunotherapy as they can amplify inside transfected cells, allowing for the sustained transgene expression over periods up to multiple weeks.¹ The durable transgene expression from replicons makes them an enticing platform to temporally control exposure of local tissues to immunological payloads and thereby maximize immune responses in the setting of vaccination or disease treatment. Toward this goal, we engineered “ON” switch replicon gene circuits responsive to the FDA-approved small molecule drug trimethoprim (TMP). In TMP-responsive circuits, regulation is imparted by a destabilizing domain (DD), derived from the Escherichia coli dihydrofolate reductase, which is targeted for degradation by proteasomes unless it is stabilized by TMP.² To regulate transgene expression, we targeted the amplification mechanisms of replicons by fusing DDs to non-structural proteins (nsPs) of the replicon backbone that are part of the replicon self-copying machinery (nsP-DD circuits) (Figure 1A). These TMP-responsive circuits were used to engineer multifunctional cancer-cell based therapeutics where living cancer cells are transfected with replicons to 1) release the immunity-driving cytokine IL-12 for a period of time and 2) upon addition of TMP, switch “on” the expression of the immunogenic cell death (ICD) mediator gasdermin D (GSDMD) to drive pyroptotic killing of the host tumor cells and release of tumor associated antigens.


Materials and Methods:: Replicon RNAs encoding the DD, IL12, GSDMD, or firefly luciferase (fLuc) as a reporter were synthesized by in vitro transcription (IVT). DD was fused to one of the four nsPs (nsP1, nsP2, nsP3, or nsP4) of the replicon backbone and/or the payload. For in vitro studies, KP lung cancer cells were electroporated with replicons using the Neon transfection system and plated in the presence or absence of 10 µM TMP, and the fLuc expression was assessed 24 h later by bioluminescence assay. For in vivo studies in mice, TMP was supplemented in the diet (ad libitum access). Replicons were injected intratumorally (i.t.) and electroporated using the ECM 830 Electro Square Porator and bioluminescence was tracked longitudinally by in vivo imaging system (IVIS) imaging. For therapeutic studies, subcutaneously implanted B16F10 melanoma tumors were treated with B16F10 cancer cells electroporated in vitro with TMP-responsive replicons encoding IL12 and GSDMD. Mice were subjected to different TMP regimens and tumor growth was monitored over time.


Results, Conclusions, and Discussions:: Among the four nsPs, fusion of DD to nsP3 along with the the payload (fLuc), provided the strongest regulation of fLuc expression in cancer cells in vitro with over 100-fold difference in the bioluminescence signal between ON (+TMP) and OFF (-TMP) states (Figure 1B). In addition, in vivo in KP tumors, the nsP3-DD/DD-fLuc inducible replicon exhibited high gene expression in the ON state with an OFF state overlapping the background control indicating non-leaky expression (Figure 1C). Implementing the nsP3-DD circuit for controlled expression of the pyroptotic protein GSDMD and the pro-inflammatory cytokine IL12, we demonstrated that intratumoral administration of replicon-transfected cancer cells reduced tumor growth when GSDMD expression was switched “on” 2 days after i.t. injection (Figure 1D). Experiments are ongoing to study the IL12 concentrations in serum and tumor, and the molecular and cellular profiles of the tumor microenvironment with different TMP regimens.

To conclude, we have successfully engineered a small-molecule responsive RNA-based platform to temporally control the expression of immunological payloads to enhance their therapeutic efficacy.




Acknowledgements (Optional): : This work was supported by the NIH (Awards EB025854 and UM1AI144462. P.Y. was supported by postdoctoral fellowships from the Ludwig Center at MIT’s Koch Institute for Integrative Cancer Research and the NIH (Award F32 AI164829-02).


References (Optional): : 1. Aliahmad, P., Miyake-Stoner, S. J., Geall, A. J. & Wang, N. S. Next generation self-replicating RNA vectors for vaccines and immunotherapies. Cancer Gene Ther (2022).

2. Iwamoto, M., Björklund, T., Lundberg, C., Kirik, D. & Wandless, T. J. A general chemical method to regulate protein stability in the mammalian central nervous system. Chem Biol 17, 981–988 (2010).