In Vivo pan CAR therapy utilizing circular RNA for treatment of autoimmune diseases

Abstract Background/ Purpose: Recently, traditional ex vivo-generated chimeric antigen receptor T (CAR-T) cell therapies have shown success in the clinic for autoimmunity indications. However, manufacturing, safety, and accessibility remain potential challenges in the ex vivo CAR-T approach. The prospect of an in vivo CAR-T cell therapy, without the need for patient cell isolation, cell culturing, and the safety risks associated with preconditioning regimens, remains a therapeutic goal. Orna Therapeutics' panCAR™ combines a synthetic, circular, coding RNA platform (oRNA®) and a proprietary immunotropic lipid nanoparticle (LNP) to drive CAR expression on the surface of immune effector cells after in vivo administration, with the potential to provide a transient, re-dosable, and scalable immune cell therapy without the need for preconditioning lymphodepletion. Methods: Anti-CD19 panCAR™ is a non-viral platform combining anti-CD19 CAR expressing oRNA with a proprietary immunotropic LNP. Anti-CD19 panCAR was evaluated for expression in T cells and depletion of target B cells in vitro and in vivo in humanized CD34+ mouse models upon intravenous injection. In a humanized mouse model of lupus, anti-CD19 panCAR was dosed four times weekly, and B cell levels and anti-double stranded DNA (anti-dsDNA) titers were evaluated. In cynomolgus macaques, anti-CD19 panCAR was dosed intravenously twice three days apart (Q3Dx2) at three dose levels (0.1, 0.5, and 1.0 mg/kg). B cell levels were then evaluated in periphery and in lymphoid tissues by flow cytometry and immunohistochemistry (IHC). Results: With anti-CD19 panCAR, high surface expression of anti-CD19 CAR was observed in a dose-dependent manner and was maintained over 96 hours on human and NHP immune cells in vitro. Furthermore, CAR-mediated cytotoxicity was observed for T cells from both species, killing CD19-expressing cell lines. In the humanized mouse model, anti-CD19 panCAR administered intravenously showed significant B cell reductions in peripheral blood, spleen, and bone marrow at 24 hours (55-95%) and sustained depletion 7 days after a single dose. The functional activity of anti-CD19 panCAR correlates with the dose level and dose number administered, demonstrating the tunability of the platform. Anti-CD19 panCAR, injected into a humanized mouse model of lupus showed complete reduction of B cells accompanied by anti-dsDNA titer reduction. By comparison, mice receiving rituximab exhibited reduced B cell numbers, but the depletion did not correlate with reduced anti-dsDNA titers. Finally, in NHPs, the cross-reactive anti-CD19 panCAR showed complete (>98%) and sustained depletion of B cells in peripheral blood at 24 hours after dosing through two weeks after the last dose, and showed robust depletion in the spleen (>99%), lymph nodes (>97%), and bone marrow (>96%) three days after the last dose. Robust anti-CD19 CAR expression was observed 24 hours after dosing. As the anti-CD19 panCAR oRNA is transient, B cell recovery is observed in the peripheral blood beginning around 2-3 weeks after treatment with full B cell recovery in tissues by Day 56. Notably, the reconstituting peripheral B cells are predominantly a naïve phenotype over a switched memory phenotype, indicating the promise of this therapy for potential immune reset. Conclusion: Orna's platform provides a non-viral, transient, tunable, and scalable approach without the need for preconditioning lymphodepletion that shows robust activity in vitro and in vivo. In NHPs, intravenous dosing resulted in deep B cell depletion in peripheral blood and lymphoid tissues. Collectively, these pre-clinical data demonstrate the potential of Orna's in vivo panCAR therapy to treat B cell-mediated autoimmunity.