B-Cell-Targeted 3DNA Nanotherapy Against Indoleamine 2,3-Dioxygenase 2 (IDO2) Ameliorates Autoimmune Arthritis in a Preclinical Model

Abstract

The tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase 2 (IDO2) has been identified as an immunomodulatory agent promoting autoimmunity in preclinical models. As such, finding ways to target the expression of IDO2 in B cells promises a new avenue for therapy for debilitating autoimmune disorders such as rheumatoid arthritis. IDO2, like many drivers of disease, is an intracellular protein expressed in a range of cells, and thus therapeutic inhibition of IDO2 requires a mechanism for targeting this intracellular protein in specific cell types. DNA nanostructures are a promising novel way of delivering small molecule drugs, antibodies, or siRNAs to the cytoplasm of a cell. These soluble, branched structures can carry cell-specific targeting moieties along with their therapeutic deliverable. Here, we examined a 3DNA nanocarrier specifically targeted to B cells with an anti-CD19 antibody. We find that this 3DNA is successfully delivered to and internalized in B cells. To test whether these nanostructures can deliver an efficacious therapeutic dose to alter autoimmune responses, a modified anti-IDO2 siRNA was attached to B-cell-directed 3DNA nanocarriers and tested in an established preclinical model of autoimmune arthritis, KRN.g7. The anti-IDO2 3DNA formulation ameliorates arthritis in this system, delaying the onset of joint swelling and reducing total arthritis severity. As such, a 3DNA nanocarrier system shows promise for delivery of targeted, specific, low-dose therapy for autoimmune disease.

Keywords: 3DNA; IDO2; arthritis; autoimmunity; indoleamine 2,3-dioxygenase 2.

Figure 1.

3DNA components and formulations. (A) Spleen cells from C57BL/6 mice were stained with unconjugated or oligo-conjugated anti-CD19 and examined by flow cytometry. Both unconjugated anti-CD19 and anti-CD19-oligo conjugate showed equal binding, demonstrating the DNA conjugation chemistry did not negatively affect the binding capacity of the antibody. (B) Purified B cells were stimulated with LPS + IL-4 to induce IDO2, transfected with anti-IDO2 siRNA, and IDO2 RNA levels were measured by qRT-PCR. Graphs show mean fold change in IDO2 ± SEM compared with control for n = 3 per condition. (C, D) With both targeting and payload moieties confirmed, 2-layer, 3DNA nanocarrier was then formulated. Representative diagrams of 3DNA targeting constructs were shown. (C) For targeting and biodistribution studies, dye-labeled oligos (red dots) are crosslinked to peripheral 3DNA sequences, while antibody-oligo conjugates (Y-shaped structures) are hybridized to peripheral 3DNA sequences. (D) For therapeutic efficacy studies, modified siRNA oligos (green) and antibody-oligo conjugates are hybridized to their respective complementary peripheral 3DNA sequences. (E) B-cell-binding capacity of 3DNA constructs was compared with the binding capacity of the unconjugated anti-CD19 antibody. B cells are able to bind antibody alone or anti-CD19 hybridized to 3DNA with identical capacity. SEM, standard error of mean.

Figure 2.

Anti-CD19-targeted 3DNA is internalized in B cells with limited binding of isotype control 3DNA. (A) Anti-CD19-3DNA or Rat Ig-3DNA hybridized with AlexaFluor488 was incubated for 60 minutes at 37°C with spleen cells from C57BL/6 mice and analyzed by imaging cytometry. Anti-CD19 3DNA targets B cells (left panel), while isotype control 3DNA does not (right panel). (B) B220 staining was used to define the cell surface based on Imagestream images. Intensity of internalized anti-CD19 or Rat Ig-3DNA staining defined as the intensity of AlexaFluor488 staining in an area defined as the cell boundary eroded toward the cell center by 4 pixels. (C) Representative images show B220 surface staining (red) and anti-CD19 or isotype control 3DNA (green) on individual cells. A representative experiment of 3 independent trials is shown.

Figure 3.

3DNA rapidly binds to target B cells in vivo. AlexaFluor647-labeled anti-CD19 or isotype control 3DNA was injected intravenously into C57BL/6 mice. Cells from the (A, B) peripheral blood and (C, D) spleen were harvested after (A, C) 10 minutes or (B, D) 30 minutes and analyzed by flow cytometry, with cell type defined with the following markers: B cells (B220), T cells (CD4 + CD8), macrophages (MΦ, CD11b), and neutrophils (GR-1). Graphs show the mean percentage of labeled cells ± SEM for n = 3 mice/group. (E) The proportion of the 2 cell types that primarily bind 3DNA, B cells, and macrophages is shown in the spleen and peripheral blood. Graphs show mean percentage of labeled cells ± SEM for n = 3 to 6 mice/group. (F) 3DNA in peripheral blood and spleen was measured by PCR. Graph shows the mean pg 3DNA ± SEM for n = 3 mice/group. Statistical significance is determined by t test with the Holm-Sidak correction for multiple comparisons. *P < 0.05; ***P < 0.001. n.s. indicates not significant; PCR, polymerase chain reaction; SEM, standard error of mean.

Figure 4.

Anti-CD19-3DNA-siIDO2 inhibits arthritis in a preclinical model. KRN.g7 mice were treated 2 times per week beginning at weaning (3 weeks of age) with (A) 9.6 µg anti-CD19-3DNA-siIDO2, anti-CD19-3DNA-siControl, Rat Ig-3DNA-siIDO2, or untreated control and (B) a dose response of siIDO2. Dose of IDO2 was determined by formulations of 0.013 to 28.3 µg siIDO2 with anti-CD19 3DNA. Anti-CD19-3DNA-siIDO2 (red) and anti-CD19-siControl (black) (9.6 µg) from panel (A) are shown again in panel (B) for comparison. Graphs show the mean ankle thickness ± SEM for n = 4 to 8 mice/group. Statistical significance was determined by 1-way ANOVA with post hoc testing with the Tukey correction for multiple comparisons. *P < 0.05. n.s., not significant; SEM, standard error of mean.