Fig. 6: Engineered circRNAs demonstrate more durable translation and functional activity in vivo.
From: Engineering circular RNA for enhanced protein production
a, CircRNA with 5% m6A incorporation encoding NanoLuc was synthesized with the following optimizations: upstream IRES topology, 5′ PABP spacer, HBA1 3′ UTR and HRV-B3 IRES with proximal loop Apt-eIF4G insertion. CircRNAs were formulated for intraperitoneal delivery in mice using CARTs. Expression was assayed using an optical imaging system after intraperitoneal injections of the fluorofurimazine substrate at the indicated timepoints. At 336 hours (14 days) after circRNA NanoLuc administration, mice were redosed. b, In vivo luminescence image of an untreated mouse (left) versus mice receiving circRNA NanoLuc (right) at 24 hours after dosing. c, Quantification of luminescence per mouse at different timepoints after circRNA NanoLuc administration. Redosing was performed at 336 hours (14 days). Data are mean ± s.e.m. for n = 3 animals per condition. d, CircRNA with 5% m6A incorporation encoding hEPO was synthesized with the following optimizations: upstream IRES topology, 5′ PABP spacer, HBA1 3′ UTR and HRV-B3 IRES with proximal loop Apt-eIF4G insertion. mRNA-encoding hEPO was synthesized with CleanCap reagent, 100% N1Ψ incorporation and a 120-nt poly(A) tail. Equimolar doses of circRNA and mRNA were formulated for intravenous delivery in mice using CARTs. Plasma hEPO was measured by ELISA in one cohort at the indicated timepoints. Reticulocytes were counted in a separate cohort at 168 hours (7 days). e, Quantification of plasma hEPO at different timepoints after circRNA hEPO or mRNA hEPO administration. Data are mean ± s.e.m. for n = 4 animals per condition. f, Plasma hEPO expression normalized to the 24-hour level of each mouse at different timepoints after circRNA hEPO or mRNA hEPO administration. Data are mean ± s.e.m. for n = 4 animals per condition. *P = 0.0487 and ***P = 0.0001 by unpaired two-sided t-test with Bonferroni correction compared to mRNA. g, Reticulocyte percentage among red blood cells at 168 hours after circRNA hEPO or mRNA hEPO administration. Data are mean ± s.e.m. for n = 4 animals per condition. **P = 0.0080 by unpaired two-sided t-test. NS, not significant. For gating strategy, see Supplementary Fig. 10b. i.p., intraperitoneal; i.v., intravenous.
