Gene therapy for immune tolerance induction in hemophilia with inhibitors

Abstract

The development of inhibitors, i.e. neutralizing alloantibodies against factor (F) VIII or FIX, is the most significant complication of protein replacement therapy for patients with hemophilia, and is associated with both increased mortality and substantial physical, psychosocial and financial morbidity. Current management, including bypassing agents to treat and prevent bleeding, and immune tolerance induction for inhibitor eradication, is suboptimal for many patients. Fortunately, there are several emerging gene therapy approaches aimed at addressing these unmet clinical needs of patients with hemophilia and inhibitors. Herein, we review the mounting evidence from preclinical hemophilia models that the continuous uninterrupted expression of FVIII or FIX delivered as gene therapy can bias the immune system towards tolerance induction, and even promote the eradication of pre-existing inhibitors. We also discuss several gene transfer approaches that directly target immune cells in order to promote immune tolerance. These preclinical findings also shed light on the immunologic mechanisms that underlie tolerance induction.

Keywords: alloantibodies; factor IX; factor VIII; gene therapy; hemophilia; immune tolerance.

Figure 1. Streamlined schematic of current and potential gene therapy based management of patients with inhibitors

Gene transfer of FVIII or FIX offers the potential of simplifying current management with a single therapeutic administration to provide both ITI and factor prophylaxis.

Figure 2. Gene therapy potentially offers several advantages over current protein ITI

A single administration (arrow) of a FVIII or FIX gene therapeutic (top panel) provides a steady exposure of antigen (blue) and increasing factor activity (red) with decreasing inhibitor titer (black). Once the inhibitor is eradicated, gene therapy provides ongoing factor prophylaxis. In contrast, protein ITI (bottom panel) requires myriad infusions during inhibitor eradication as well as ongoing administrations for maintenance of tolerance.

Figure 3. A simplified illustration of the cellular compartments responsible for the persistence of inhibitors

Effective cooperation between Factor-specific CD4+ T-cells and Factor-specific memory B-cells leads to the differentiation and proliferation of the latter to form inhibitor-producing plasma cells. This cooperation requires the expression of T-cell receptors (TCR) that recognize Factor-peptides presented by MHC class II as well as effective co-stimulatory interactions, such as B7 (CD80/86) binding with CTLA-4. The presentation of factor by professional Antigen Presenting Cells (APC) facilitates that activation of these T-cells, which in turn, activates additional B-cells increasing inhibitor secretion; conversely, the activation of factor-specific Treg-cells impedes this process through the production of inhibitory cytokines including IL-10. As detailed in the text, reductions in Factor-specific B- and T-cells and increased Factor-specific Treg-cells contribute to inhibitor eradication and tolerance induction.

Figure 4. Inhibitor eradication after FVIII gene therapy is associated with an increase in Treg-cells

(A) As previously reported [59], liver-directed cFVIII gene therapy for a HA dog (K01) with a pre-existing inhibitor resulted in the disappearance of both the inhibitor-titer and FVIII-specific immunoglobins; this eradication coincided with an increase in the percentage of CD25+FoxP3+, CD4+ positive cells, assumed to be Treg-cells, from baseline. Gene therapy also tolerized K01, as demonstrated by the unchanged inhibitor-titer and FVIII-specific immunoglobin levels after protein challenge (dotted vertical line). This exposure of exogenous FVIII protein was, however, associated with a rebound of Treg-cells. (B) A similar spike in Treg-cells was seen in two other HA dogs (K03, L44) with pre-existing inhibitors, but not in HA dogs without inhibitors (M06, Linus, L51) [57, 93].

Figure 5. Inhibitor eradication after FIX gene therapy is associated with an increase in anti-inflammatory cytokines

As previously reported [67], liver-directed FIX gene therapy results in inhibitor eradication and immune tolerance induction for a HB dog (Wiley). The amnestic rise of the inhibitor is associated with a reactive increase in IL-10 levels, which slowly decreases with increasing FIX activity levels. IL-10 is an inhibitory cytokine produced by Treg-cells. This tolerance was maintained despite protein challenge (dotted vertical line), though it was associated with a rebound in IL-10 levels.