Background: While CAR-T therapy has revolutionized the treatment of hematologic malignancies and shown promise for several autoimmune disorders, its widespread clinical application is hindered by the adverse effects of lymphodepleting, logistic complexity, prohibitive costs and safety concerns regarding viral vectors integration. Emerging RNA-based in vivo CAR-T cell engineering approaches offers an alternative for addressing these challenges. Leveraging Therorna’s proprietary circRNA platform, we developed TI-0032, a novel anti-CD19-CAR circular RNA therapeutic delivered by targeted lipid nanoparticles (tLNPs). By combining its convenient accessibility, stable durability and high efficiency, TI-0032 holds significant potential to overcome the limitations of traditional ex vivo CAR-T therapies, enabling broader clinical applicability in both hematologic and autoimmune conditions.

Objectives: To evaluate the therapeutic potential of TI-0032 through comprehensive in vitro systems, as well as in vivo humanized mouse and non-human primate (NHP) models.

Methods: TI-0032 comprises a stable circRNA payload encapsulated in tLNP formulated with a novel ionized lipid. The surface of tLNP is engineered with an anti-CD8 humanized antibody fragment linked through a unique conjugation site, enabling specific and high efficient targeting and expression in CD8+T cells. We utilized multiple in vitro systems to assess targeting specificity, T cell activation, and autologous B cell depletion. In vivo pharmacodynamics were evaluated in humanized mice (using TI-0032) and cynomolgus monkeys (using TI-0032 surrogate) via flow cytometry, immunohistochemistry (IHC), and RNA-seq.

Results: TI-0032 demonstrates the robust physiochemical attributes resulting from our rational design and optimized production process. As a complex pharmaceutical formulation, it exhibited exceptional stability, maintaining consistent structural integrity and functional activity even after multiple freeze-thaw cycles. Crucially, in non-activated primary T cells, TI-0032 mediated CAR expression in over 90% CD8+T cells, which persisted for over two weeks, underscoring the platform’s capability for long-term stability. Functional in vitro assays in non-activated PBMCs demonstrated that TI-0032 induced specific, dose-dependent CAR expression in CD8+ T cells, triggering robust activation and proliferation. Importantly, TI-0032 treatment achieved complete elimination of B cells in PMBCs derived from both health donors and patients with Systemic Lupus Erythematosus (SLE). In a humanized NOG-PBMC model, a single dose of TI-0032 resulted in complete B cell depletion in both peripheral blood and spleen within 24 hours. Furthermore, in humanized NOG-PBMC mice bearing Nalm6-luc tumors, three doses of TI-0032 at sub-microgram level conferred complete tumor remission. In cynomolgus monkeys, peripheral blood B cells were depleted completely as early as 6 hours post the first dose of the TI-0032 surrogate. Splenic B cells were eradicated after three doses, as confirmed by both IHC and RNA-seq.

Conclusions: TI-0032 represents a transformative in vivo CAR-T modality that harnesses the unique stability and durability of circRNA to enable robust therapeutic activity. These results support further clinical development of TI-0032 for B-cell malignance and autoimmune diseases. Combined with its off-the-shelf availability, scalable manufacturing, and precise T-cell targeting, TI-0032 presents a promising opportunity of unleashing the full potential of CAR-T technology.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: None declared.