RIG-I is involved in regulating the DNA-induced STING degradation.

<p>(A) Western blotting analysis of RIG-I expression after MRC-5 cells were transfected with pcDNA3.1. (B) Time course assessment of RIG-I mRNA after MRC-5 cells were transfected with pcDNA3.1 (0.05μg) for the indicated time points. (C-G) Western blotting shows a reverse correlation between STING and RIG-I under different conditions: dose (C), time (D, E), DNA types (F), and Bortezomib treatment (G). (H) Silencing RIG-I partially reverses the STING degradation. (I) qRT-PCR assessment of RIG-I expression after STING knockdown in MRC-5 cells. Asterisk (*), P <0.05.</p

Proteasome-mediated degradation plays a role in STING reduction.

<p>(A) Time course assessment of STING mRNA expression after MRC-5 cells were transfected with pcDNA3.1 (0.05μg). (B) Western blotting assessment of STING after MRC-5 cells were transfected with pcDNA3.1 (0.05μg) with treatment of Bortezomib (20 nM) or DMSO for 18h. (C)Western blotting assessment of immunoprecipitated STING with anti-ubiquitin antibody.</p

STING degradation occurs in HDCs but not in HEK293.

<p>(A) Western blotting assessment of STING and RIG-I in MRC-5, 2BS, KMB17 and hUC-MSC cells after transfection with 0.5 μg of pcDNA3.1 for 24 h. (B) MRC-5 and HEK293 cells were transfected with 0.5 μg of pcDNA3.1 for 24 hours. The indicated molecules were assessed by western blotting. (C, D) qRT-PCR assessment of mRNA levels (C) and ELISA assessment of protein levels (D) of IL-6 in MRC-5 and HEK293 cells after transfection of pcDNA3.1. (E) ULK1 S556 phosphorylation was assessed by western blotting in MRC-5 and HEK293 cells. Asterisk (*), P <0.05.</p

The plasmid DNA induces STING protein reduction in MRC-5 cells.

<p>(A) After 48 h transfection with different concentrations of pcDNA3.1, the MRC-5 cell lysates were analyzed by immunoblotting with anti-STING. (B, C) Time course assessment of STING expression induced by DNA. (D) Western blotting assessment of STING after 48 h transfection of MRC-5 cells with equal amount of circular plasmid pcDNA3.1, plasmid vector PGL-3, liner plasmid pcDNA3.1, 2 kb PCR DNA fragment amplified from pcDNA3.1, genomic DNA derived from MRC-5 cells, DNA virus (rTv-Fluc and HSV-2).</p

STING serves as a major mediator for the plasmid DNA-induced IFN-β response.

<p>(A) Analysis of the dose effect on IFN-β production induced by DNA transfection. MRC-5 cells were transfected with pcDNA3.1 with indicated amounts for 6 hours. IFN-β mRNA was analyzed by qRT-PCR. (B) Time course assessment of IFN-β induction by pcDNA3.1 (0.05μg). (C, D) After 24 h transfection of MRC-5 cells with pcDNA3.1, cells were infected with rTV-Fluc (C) or Sendai virus (D) for another 24 hours before a luciferase report assay for rTV-Fluc (C) and qRT-PCR for Sendai virus (D). (E) Western blotting showing STING knockdown by siRNA after 72 h transfection in MRC-5 cells. (F) qRT-PCR assessment of IFN-β after STING was knockdown in MRC-5 cells that were transfected with pcDNA3.1(0.2μg). (G) A luciferase report assay showing rTV-Fluc virus replication after STING knockdown. Data are presented as mean ± SD (n = 3). Asterisk (*), P <0.05.</p

IL-6 and RIG-I contribute additively to the DNA-induced STING degradation.

<p>(A) qRT-PCR assessment of expression of indicated interleukins after MRC-5 cells were transfected with pcDNA3.1 (0.05 μg) for 9 hours. (B) ELISA analysis of IL-6 protein levels in the supernatants of cells at different time points. (C) qRT-PCR analysis of IL-6 mRNA at different time points. (D) Western blotting assessment of STING expression after MRC-5 cells were treated with IL-6R antibody with pcDNA3.1 transfection. (E) Western blotting assessment of STING expression after application of IL-6R antibody and siRIG-I. (F) qRT-PCR (up) and ELISA analysis (down) of IL-6 expression after STING knockdown. Asterisk (*), P <0.05.</p

RIG-I or IL-6 promotes STING degradation by activating ULK1 in MRC-5 cells.

<p>(A) Western blotting assessment of the levels of ULK1 S556 phosphorylation triggered by dsDNA. (B) Western blotting assessment of the ULK1 S556 phosphorylation in MRC-5 cells with RIG-I knockdown or IL-6R antibody treatment.</p

Codon-optimized hFVIII to induce tolerance and correction.

<p>BALB/c-HA and BL/6-129/sv-HA mice were injected with 10¹¹vg/mouse of an AAV8 vector expressing codon-optimized hFVIII. <b>A.</b> Coagulation times (aPTT in sec) and <b>B.</b> HFVIIII activity were measured as a function of time after vector administration. Data are averages ±SD for n = 4/group. Mice were challenged (starting at week 10 after gene transfer) with hFVIII at the same dose and schedule as in previous experiments. Anti-hFVIII formation was measured <b>C.</b> as HFVIIII-specific IgG1 titers, and <b>D.</b> by Bethesda assay. Values in panels C and D are shown for individual animals and as averages ± SD and plotted on the same scale as in Fig. 2 to compare magnitude of responses.</p

Anti-CD20 treatment to prevent antibody formation in hFVIII protein replacement therapy.

<p>BL/6-129/sv-HA and BALB/c-HA mice were treated with αCD20 antibody as outlined in Fig. 1A (indicated with large arrows) followed by 4 weeks of hFVIII challenge (indicated by small arrows) beginning at 4 weeks after the second αCD20 administration. Mice were treated with hFVIII twice more following the same schedule. Antibody formation against HFVIIII was measured by Bethesda assay (<b>A</b>) and anti-hFVIII IgG1 ELISA (<b>B</b>) two weeks after each 4-week challenge. Control mice did not receive αCD20. Gray triangles represent B cell recovery. Data are averages ±<u>SD</u> for n = 3–5/group.</p

Depletion of B cells using anti-CD20.

<p><b>A.</b> Treatment schedule. Hemophilia A (HA) mice in “αCD20” groups received a dose of 10 mg/kg IgG2a αCD20 i.v. on day 0 and day 21. One week following the first αCD20 injection, mice in “AAV8” groups received 10¹¹ vg/mouse of AAV8-hFVIII. Blood samples were collected at indicated time points. Mice were challenged with intravenous hFVIII (1 IU/mouse, weekly for 4 weeks) at indicated time points. <b>B.</b> Representative examples of B cell depletion in different lymphoid organs of BALB/c-HA mice 1 day and 7 weeks after the second injection second αCD20 injection. Numbers in each histogram represent percent CD19⁺ lymphocytes (as shown by forward and side scatter gating in left panel) for both untreated control (in black) and αCD20-treated animals (red). Cells were stained with anti-CD19 antibody conjugated to V450 fluorochrome at 1 day post second injection, or to APC-Cy7 for the 7-weeks post-αCD20 time point.</p