A Novel Platform for Immune Tolerance Induction in Hemophilia A Mice

Abstract

Hemophilia A (HA) is an X-linked bleeding disease caused by factor VIII (FVIII) deficiency. We previously demonstrated that FVIII is produced specifically in liver sinusoid endothelial cells (LSECs) and to some degree in myeloid cells, and thus, in the present work, we seek to restrict the expression of FVIII transgene to these cells using cell-specific promoters. With this approach, we aim to limit immune response in a mouse model by lentiviral vector (LV)-mediated gene therapy encoding FVIII. To increase the target specificity of FVIII expression, we included miRNA target sequences (miRTs) (i.e., miRT-142.3p, miRT-126, and miRT-122) to silence expression in hematopoietic cells, endothelial cells, and hepatocytes, respectively. Notably, we report, for the first time, therapeutic levels of FVIII transgene expression at its natural site of production, which occurred without the formation of neutralizing antibodies (inhibitors). Moreover, inhibitors were eradicated in FVIII pre-immune mice through a regulatory T cell-dependent mechanism. In conclusion, targeting FVIII expression to LSECs and myeloid cells by using LVs with cell-specific promoter minimized off-target expression and immune responses. Therefore, at least for some transgenes, expression at the physiologic site of synthesis can enhance efficacy and safety, resulting in long-term correction of genetic diseases such as HA.

Keywords: Tregs; gene therapy; hemophilia A; inhibitor titers reversion; targeted FVIII expression.

Figure 1

Endothelial- and Myeloid-Specific Transgene Expression in the Liver of LV-Injected Mice (A–E) GFP expression in the liver and the spleen of mice injected with LV.VEC-GFP-miRT-122–142 (n = 9) (A), LV.VEC-GFP (n = 9) (B), LV.VEC-GFP-miRT-142-3p (n = 9) (C), or LV.CD11b-GFP ± miRT-126 (n = 9) (D and E) 1 week and 3 months after injection. After injection of LV.VEC-GFP, transgene expression was observed in LSECs and splenic endothelial cells, with some off-targets in hepatocytes (white arrowheads) and myeloid cells (white arrows) (B). The addition of the miRT-142-3p avoided transgene expression in hematopoietic cells but not in hepatocytes (C). The addition of the miRT-122–142-3p restricted GFP expression to endothelial cells without off-targets in other cell types, as shown by the co-staining of the endothelial marker Lyve-1(red) with the GFP (green) (A). After injection of LV.CD11b-GFP, transgene expression was observed mainly in hepatic and splenic myeloid cells, with few GFP-expressing endothelial cells (white arrows) (E); by adding miRT-126 to the construct transgene, expression was detected only in myeloid cells, as shown by co-staining for the macrophage marker F4/80 (red) and GFP (green) (D). The scale bars represent 25 μm.

Figure 2

Long-Term hFVIII Expression in HA Mice after Injection of LV.VEC-hFVIII ± miRT-122–142-3p and LV.CD11b-hFVIII ± miRT-126 (A–H) HA mice were injected for the expression of hFVIII in endothelial cells with the LV containing the endothelial-specific VEC promoter with (n = 9) or without (n = 5) the addition of miRT-122–142-3p (A, C, E, and G) or in myeloid cells with the LV containing the myeloid-specific CD11b promoter with (n = 5) or without (n = 5) the addition of miRT-126 (B, D, F, and H). (A, B, E, and F) Graphs showing the mean percentages ± SD of hFVIII activity in plasma of HA mice up to 1 year after LV injection. Mice injected with VEC-hFVIII-122–142 (E) or CD11b-hFVIII-126 (F) were immunized with a subcutaneous injection of hFVIII in incomplete Freund’s adjuvant (IFA) (A, B, E, and F: p < 0.001 compared with control HA group). (C, D, G, and H) ELISA showing the presence of anti-FVIII antibodies in plasma of LV injected mice (means ± SD). No anti-FVIII antibodies were detected in plasma of HA mice injected with LV.VEC-hFVIII ± miRT-122–142-3p (C and G) or CD11b-hFVIII-126 (H). Thirty percent of HA mice injected with LV.CD11b-hFVIII developed anti-FVIII antibodies between 8 and 12 weeks after LV injection (D). Plasma dilution was 1:2,000. Ctr+, pooled plasma from LV.PGK-hFVIII-injected mice that developed anti-FVIII antibodies.

Figure 3

hFVIII Expression in Hepatic and Splenic Endothelial Cells and Myeloid Cells of HA Mice 1 Year after LV Injection (A–F) Immunohistochemistry showing hFVIII expression in hepatic and splenic endothelial cells after injection of LV.VEC-hFVIII-122–142 (A and B) and in hepatic and splenic myeloid cells after injection of LV.CD11b-hFVIII-126 (C and D). Control liver and spleen from HA mice showed no hFVIII positive staining (E and F). The scale bars represent 100 μm.

Figure 4

hFVIII Activity after LV.VEC-hFVIII ± miRT-122–142-3p Injection in HA Mice Previously Immunized with hFVIII HA mice were immunized with FVIII, and 4 weeks later injected with LV.VEC-hFVIII ± 122–142. (A and B) hFVIII activity in plasma of mice injected with LV.VEC-hFVIII (A) and LV.VEC-hFVIII-122–142 (B) increased over time, reaching 5%–6% FVIII activity, and remained stable up to 1 year. Rechallenge with hFVIII had no effects on hFVIII activity in plasma of both groups of mice (A and B: p < 0.001 compared with control group). (C) BU present in plasma of immunized mice decreased over time after injection of LV.VEC-hFVIII ± 122–142. (D–F) Line-graphs showing anti-FVIII IgG1 (D), IgG2a (E), and IgG2b (F) antibody titers in plasma of FVIII-immunized HA mice injected with LV.VEC-hFVIII and LV.VEC-hFVIII-122–142 (C–F: p < 0.001 compared with control group). Indicated are means ± SD.

Figure 5

GFP Expression in Hepatic and Splenic Plasmacytoid Dendritic Cells after Injection of LV.CD11b-GFP ± miRT-126 (A and B) FACS analysis showing GFP expression in pDCs (CD11c⁺, B220⁺, pDCA-1⁺) and in DCs (CD11c⁺, pDCA-1⁻) from liver (A) and spleen (B) of mice injected with LV.CD11b-GFP ± miRT-126. Mice injected with the LV.PGK-GFP were used as controls. The addition of miRT-126 to the LV.CD11b-GFP drastically reduced the GFP expression in hepatic and splenic pDCs. Indicated are mean percentages ± SD (n = 6 or 7 per group).

Figure 6

Comparison of Different Forms of BDD-hFVIII HA mice (n = 3–5) were injected with LV containing the VEC promoter driving expression of BDD-hFVIII ± miRT-122–142, RH, and co-hFVIII-miRT-122–142. (A) Two weeks after LV injection, FVIII activity in plasma of injected mice showed an average of 4% for BDD-hFVIII ± miRT-122–142 and RH, and 7% for co-hFVIII-miRT-122–142. Although hFVIII activity remained stable to 5% in plasma of mice injected with LV.VEC-hFVIII ± miRT-122–142, LV.VEC-RH-, and LV.VEC-co-hFVIII-miRT-122–142-injected HA mice showed an average of 8% and 11%–12% of hFVIII activity, respectively, and remained stable up to 52 and 36 weeks after injection, and immunization with FVIII did not affect the FVIII activity in plasma of these mice (p < 0.001 for LV.VEC-cohFVIII-122–142 versus LV.VEC-hFVIII ± 122–142). (B) ELISA showing the absence of anti-FVIII antibodies in plasma of mice injected with the different LVs. Immunization with FVIII did not result in anti-FVIII antibody production in LV-injected mice. Plasma dilution was 1:2,000. Ctr+, pooled plasma from LV.PGK-hFVIII-injected mice that developed anti-FVIII antibodies. (A and B) Indicated are means ± SD. (C) Blood loss (mean ± SD) was evaluated 24 weeks after LV injection by measuring the absorbance at 597 nm (A_597nm) of blood content in the collection tubes by lysis of red blood cells. (D) Tail bleeding times (mean ± SD) were assessed by monitoring the blood flow into collection tubes containing saline solution, and times to stop bleeding were recorded. **p < 0.01; ***p < 0.001.

Figure 7

Effects of Treg Depletion after Gene Therapy with LV.VEC-hFVIII ± 122–142 (A) hFVIII activity in plasma of LV-injected mice reached a level of 4% with LV.VEC-hFVIII ± 122–142 (n = 4 or 5). FVIII activity remained stable up to 10 weeks after LV injection. hFVIII activity started to decrease 3 weeks after Treg depletion, reached almost undetectable levels, and increased again 16 weeks after Treg depletion, returning to levels observed before Treg depletion 4 weeks later (p < 0.001, treated mice compared with control HA group). (B) Bethesda units (BU) started to be detected 3 weeks after Treg depletion and reached the peak 12 weeks after Treg depletion, and then BU decreased again 8 weeks later. (C–E) ELISA showed no anti-FVIII antibodies in plasma of LV-injected mice up to 14 weeks after LV injection. Three weeks after Treg depletion, the presence of anti-FVIII IgG1 (C), IgG2A (D), and IgG2b (E) was detected in the plasma of treated mice. Although anti-FVIII IgG2a (D) and IgG2b (E) titers remained high, anti-FVIII IgG1 (C) titers decreased concomitantly with BU 12 weeks after Treg depletion. (A–E) Indicated are means ± SD. (F) Blood loss (mean ± SD) was evaluated at different times after LV injection by measuring the absorbance at 597 nm (A_597nm) of blood content in the collection tubes by lysis of red blood cells. (G) Tail bleeding times (mean ± SD) were assessed by monitoring the blood flow into collection tubes containing saline solution, and times to stop bleeding were recorded. n = 3 or 4 per group. **p < 0.01; ***p < 0.001.

Figure 8

Endothelial-Specific FVIII Expression in Different HA Mouse Strains (A–D) BALB/c HA mice (n = 4) and B6/129 HA mice (n = 5) were injected with LV.VEC-hFVIII and hFVIII activity (A and C), and eventual presence of anti-FVIII antibodies (B and D) was evaluated starting from 2 weeks after injection. hFVIII activity in the plasma of LV-injected mice reached 5%–6% up to 28 weeks after injection in BALB/c HA mice (A), and the same activity was detected in plasma of treated B6/129 HA mice up to 24 weeks after injection. (B and D) ELISA showing no anti-FVIII antibodies in the plasma of injected BALB/c and B6/129 HA mice up to 28 and 24 weeks, respectively, after injection. Plasma dilutions were 1:200 and 1:2,000. Ctr+, pooled plasma from LV.PGK-hFVIII-injected mice that developed anti-FVIII antibodies. (A–D) Indicated are means ± SD. (E) Blood loss (mean ± SD) was evaluated 28 weeks and 20 weeks after LV injection in BALB/c-HA and B6/129-HA mice, respectively, by measuring the absorbance at 597 nm (A_597nm) of blood content in the collection tubes after lysis of red blood cells. (F) Tail bleeding times (mean ± SD) were assessed by monitoring the blood flow into collection tubes containing saline solution, and times to stop bleeding were recorded. n = 3–5 per group. **p < 0.01; and ***p < 0.001.