Delivery of Circular RNAs into Splenic Immune Cells via Intravenous Administration of Polyaspartamide Derivative Polyplexes

<i>In vivo</i> chimeric antigen receptor (CAR) cell engineering, such as CAR T, induces CAR expression in target immune cells via the systemic administration of CAR constructs. This study investigated polyplex formation with circular RNAs to evaluate its systemic delivery efficacy into the spleen, where an abundance of various immune cells resides. Firefly luciferase (FLuc)-coded circular RNAs with Coxsackievirus B3 internal ribosome entry site (IRES) were synthesized via end-to-end self-targeting and splicing reaction. A cationic polyaspartamide derivative, PAsp(DET/CHE), which enhances endosomal escapability and optimizes hydrophobicity for particle stability, has been utilized to formulate linear or circular RNAs. PAsp(DET/CHE) successfully formulated circular RNAs into polyplexes with physicochemical characteristics similar to linear RNA polyplexes, and their polyplexes significantly improved RNA delivery efficacy in various cultured immune cells. PAsp(DET/CHE) polyplexes achieved spleen-targeted delivery of linear and circular RNAs after intravenous administration, regardless of RNA payloads, and induced high FLuc expression for up to 48 h. Notably, the circular RNA-loaded polyplexes exhibited higher T cell delivery efficacy with relatively lower accumulation in dendritic cells and macrophages than the linear RNA-loaded polyplexes. Therefore, the optimized polyplex formulation with circular RNAs can be utilized as an <i>in vivo</i> CAR RNA delivery platform for future T cell therapies.