Introduction: Typical gene delivery systems include viral vectors, lipid nanoparticles, and cationic polymers, designed to facilitate the transfection across a broad range of cell types. The primary considerations for these gene delivery systems include cationic toxicity and innate immune responses triggered by the delivery vehicle. In the context of cancer in situ vaccination, we envisioned that developing a non-viral mRNA vector capable of inducing immunogenic cell death (ICD) can reinterpret the traditional cytotoxicity drawback of gene delivery system as a mechanism for immune priming, allowing the delivered mRNA to be translated before programmed cell death occurs. For therapeutic application, we used cytokine mRNA, especially IL-12 mRNA, to leverage cancer cell as cytokine and neoantigen reservoir. Meticulous mechanisms of ICD and gene delivery were also elucidated, revealing that ICD induction and gene transfection efficiency is depend on the secondary structure of artificial polypeptide
Materials and
Methods: We designed quaternary amine-based helical polypeptides modified with guanidine moiety. As quaternary amine-based polypeptide disrupts the phospholipid bilayer, we speculated that the guanidine moiety, which enhances the interaction with phosphate groups of phospholipids and nucleic acids, could improve the polypeptide. To optimize the ratio of quaternary amine and guanidine moiety, various ratios (1:0, 3:1, 1:1, 1:3, 0:1) were tested by the ability of ICD induction and polyplex formation and gene transfection. For in vivo stabilizing agent, hyaluronic acid (HA) was exploited. in vitro mechanism study was explored with L-polypeptide and D,L-polypeptide to elucidate the effect of helical structure of polypeptide on ICD and mRNA transfection. in vivo study was conducted with MC38 mouse cancer model to study in vivo luciferase transfection and in vivo anticancer efficacy.
Results, Conclusions, and Discussions: In this study, we tried to reinterpret the chronic issue of cytotoxicity in gene delivery systems as a means to activate cancer adaptive immunity. To achieve this, we developed a polypeptide-based mRNA carrier, PG1T3, which can induce ICD in tumor cell and optimized its ratio of guanidine, enhancing interaction with phospholipids, and triethyl ammonium, disrupting the cell membrane. Notably, we discovered for the first time that the secondary structure of polymers can influence their ICD-inducing ability and examined how the cellular stress, such as mitochondrial dysfunction and ER stress, affects the release of DAMPs. As PG1T3 cannot be protonated like conventional gene delivery systems, the mechanism how polyplex delivers the mRNA to the cytosol was also elucidated. Delivering IL-12 mRNA, PG1T3 makes cancer cells produce cytokines during the early stages of transfection and undergo ICD within 3 days. Furthermore, we validated the efficacy of PG1T3 in vivo and found that hyaluronic acid (HA) coating could enhance its performance. In conclusion, this study not only proposes an optimal mRNA delivery system for eliciting antitumor immunity but also provides a detailed mechanism of how membrane-disrupting polymers deliver mRNA and induce ICD.
