Inhibition of IFNAR-JAK signaling enhances tolerability and transgene expression of systemic non-viral DNA delivery

Figures

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  Figure 1. Systemic delivery of DNA by LNPs induces significant acute cytokine responses C57BL/6 mice (n = 5) were intravenously (i.v., tail vein) dosed with LNP formulations encapsulating (A) 5 μg plasmid DNA (DNA-LNP); (B) 5 μg plasmid DNA or mRNA (mRNA-LNP); (C) 25 μg closed-ended linear duplex DNA (CELiD-LNP) or 25 μg plasmid DNA (DNA-LNP); and (D) 5 μg CpG-containing or CpG-depleted plasmid DNA. Cytokine and chemokine levels in plasma were measured at 3–4 h or 20 h post-dosing. Data shown as log2 fold increase over baseline levels (naive mice) in radar plot and as geometric mean with 95% CI in bar graphs; naive levels are shown as dashed line for each cytokine in bar graphs.

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  Figure 2. Ablation of the cGAS-STING, but not the TLR9, TLR2, MyD88, or inflammasome pathways, reduced pro-inflammatory cytokine levels and improved survival after systemic dosing of DNA-LNP Wild-type (WT) and various innate-immunodeficient knockout (KO) mice (n = 5/group) were i.v. (tail vein) dosed with LNP formulation encapsulating CpG-depleted or CpG-containing hFIX transgene (DNA-LNP). (A–F) Levels of various pro-inflammatory cytokines and chemokines in WT and KO mice were measured by Luminex 4 h post-dosing with 5 μg DNA-LNP. Data represented as log2 fold increase over baseline levels (naive mice). Survival of mice in each group was followed out to 14 days post-dosing with 25 μg (H) or 50 μg (G and I) dose of DNA-LNP. Survival data were analyzed by log rank (Mantel-Cox) test; ∗∗p < 0.001; ns, not significant.

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  Figure 3. Targeting Janus kinase improves tolerability of DNA-LNP WT and STING-deficient Goldenticket (STING(Gt)) mice (n = 5/group) were i.v. dosed via tail vein with 50 μg hFIX transgene encapsulated in LNP. WT mice were either untreated or treated orally with 200 μg/dose of Janus kinase (JAK) inhibitors (ruxolitinib or baricitinib) four times (30 min prior to and days 1, 2, and 3 post-DNA-LNP dosing). Plasma cytokine levels were measured at 4 h post-dosing in WT mice treated with either (A) ruxolitinib or (B) baricitinib. Data represented as log2 fold increase over baseline levels (naive mice). (C) Survival of mice in each group was followed out to 30 days. (D) hFIX protein level in blood plasma of all groups was measured by ELISA at weeks 2 and 4 post-dosing. Survival data were analyzed by log rank (Mantel-Cox) test, and hFIX data was analyzed using ordinary one-way ANOVA with Tukey’s post hoc tests for multiple comparisons at each time point; ∗∗p < 0.001; ns, not significant.

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  Figure 4. Targeting the type I interferon pathway improves DNA-LNP tolerability and transgene expression WT, IL-6 KO, IFNAR KO and IFN-γ KO mice (n = 5/group) were i.v. dosed via tail vein with LNP formulation encapsulating 25–50 μg hFIX transgene (DNA-LNP). (A and B) Survival for each group of mice was followed out to 10–30 days post-dosing and analyzed by log rank (Mantel-Cox) test; ∗p < 0.01; ns, not significant. (C–E) WT mice were either untreated or treated once intraperitoneally (i.p.) with 300 μg/dose of anti-mouse IFNAR antibody (aIFNAR) 3 h prior to dosing with 25 μg DNA-LNP on day 0. (C) Plasma cytokine levels measured at 4 h post-dosing are represented as log2 fold increase over baseline levels in naive mice. (D and E) WT mice were redosed with 50 μg DNA-LNP at day 41 and (D) survival was followed out to 70 days post-dosing and analyzed by log rank (Mantel-Cox) test; ∗p < 0.01; ns, not significant. (E) hFIX levels in blood plasma were measured by ELISA at the indicated time points for all surviving mice and analyzed using ordinary one-way ANOVA with Tukey’s post hoc tests for multiple comparisons at all time points, with ∗p < 0.01. (F) WT mice were treated with 300 μg/dose of…