Temporal Immunomodulation via Methylation Epigenetics Counteracts Immune Evasion by Expanding the Time Window

Epi-immunotherapy holds substantial potential in overcoming intrinsic and acquired resistance in tumor immunotherapy. However, most epi-immunotherapy strategies fail to match the dynamics of immune responses due to a lack of temporal regulation, resulting in short-lived efficacy. In addition, the instability and inefficient intracellular delivery of gene regulatory drugs, especially siRNA, further limit the therapeutic efficacy of epi-immunotherapy. Here, we propose and implement a temporal immunomodulation via methylation epigenetics (TIME) strategy through the combined intravenous administration of the FDA-approved small-molecule drug azacitidine and a BP/siPRMT1 nanocomplex. Azacitidine serves as the "Ignite" module, while BP/siPRMT1 was prepared by simple mixing of chemically synthesized boronated polypeptide (BP) with siPRMT1 at room temperature and functions as the "Sustain" module. By rapidly reducing intratumoral methylation levels through azacitidine, the expression of major histocompatibility complex class I (MHC-I) and secretion of interferon gamma (IFN-γ) were significantly enhanced, thereby igniting a hot tumor microenvironment. Subsequently, BP/siPRMT1 maintained high levels of MHC-I expression and IFN-γ secretion, sustaining the azacitidine-ignited antitumor immunity. Notably, the TIME strategy increased the proportion of MHC-I-positive tumor cells by 6.7-fold in tumor tissues. This "Ignite-Sustain" feature enables rapid immune sensitization and long-term immune maintenance. Compared to the delivery of an epi-drug or nucleic acid drug alone, the TIME strategy effectively expands the critical time window for immune activation, achieving durable and controllable tumor immunotherapy.