Strategies to Prevent or Reverse Immune Responses Against Factor In Gene Replacement for Hemophilia B.
Authors: Nayak Sushrusha , Moghimi Babak , Herzog Roland W 
Published: October 2019

Abstract
Abstract 3761
Factor IX (F.IX) deficiency resulting from large gene deletions or other null mutations can have severe ramifications due to the lack of F.IX immune recognition as a self-protein in hemophilia B. Hence, therapeutic treatment with F.IX protein can result in inhibitory antibody formation, thereby negating therapy. Muscle-directed gene therapy is an attractive novel treatment modality because of the safety and simplicity of vector administration but is hampered by an increased risk of such immune responses. Previously, we demonstrated that the drug cocktail of immune suppressants Rapamycin (Rapa), IL-10 and a specific peptide (encoding a dominant CD4+ T cell epitope) caused an induction of Treg with a concomitant antigen specific apoptosis of Teff (J. Thromb. Haemost. 7:1523, 2009). This protocol was effective in preventing inhibitor formation against human F.IX upon subsequent IM administration of an AAV-hF.IX vector in C3H/HeJ hemophilia B mice with targeted F.IX gene deletion. Here, we used this model to test whether an inhibitor response to gene therapy can be reversed. IM injection of the AAV1-hF.IX vector resulted in a high-titer inhibitory antibody response of on average 8 BU (8 mg IgG1/ml plasma) within 1 month. Transduced mice had aPTTs of ≂f80 sec and lacked systemic hFIX expression. Subsequently, the animals (n=5) were treated with IP injections of rapa/IL-10/FIX peptide 3 times/week for 4 weeks, resulting in a significant reduction of inhibitor titers to <2 BU by 1 month after tolerization. Unresponsiveness was maintained for >5 months. This reduction in the inhibitors was accompanied by a drastic decrease in IgG1 response and an increase in hF.IX production from 0 ng/ml before tolerization to a stable level of 200–650 ng/ml. Clotting times gradually declined to 58–62sec, representing a coagulation activity of ≂f1% of normal. In contrast, transduced control mice that were not treated with the tolerance protocol failed to show hF.IX expression or correction of aPTTs and maintained inhibitors for the duration for the experiment. A problem with prophylactic use of this protocol against inhibitor formation is that the specific T cell epitopes may not be known in a patient. However, when we used hF.IX protein instead of the peptide, the tolerance protocol was not effective and thus required further optimization. In humans, rapamycin is mostly given in oral form. We compared IP, SQ, IV, and oral routes of drug administration in a defined T cell receptor transgenic model (DO.11.10 Rag-/- mice). Induction of ovalbumin-specific CD4+CD25+FoxP3+ Treg up to 20% of CD4+ T cells was achieved with all these routes, except that oral administration of rapa was less effective. We reasoned that more frequent oral rapa administration may overcome this limitation. After additional optimization steps, we tested the following protocol. Rapa was given orally on a daily schedule for 1 month to the hemophilia B mice, during which time a subtherapeutic dose of hF.IX protein (0.1 IU) was given IV twice/week. Vector was given IM 1 week before the end of the tolerance protocol. This regiment was effective and prevented inhibitor formation (<1 BU), resulting in systemic hF.IX expression. We propose that alternative protocols based on use of rapamyin and either the entire cognate protein or a known T cell epitope (which can be mapped during the course of an immune response) can applied to prevent or reverse inhibitor responses in gene therapy for hemophilia.

Disclosures:
Herzog: Genzyme Corp: Patents & Royalties.