Hepatic Tolerance Induction Prevents Inflammatory Responses To Factor IX Expressing Cells at Supplementary Sites of Gene Transfer.
Authors: Dobrzynski E. , Mingozzi F. , Wang L. , Mingle B. , Cao O. , Herzog R. W. 
Published: October 2019

The use of gene replacement therapy is an attractive approach for the treatment of the genetic bleeding disorder hemophilia B (caused by mutations in the coagulation factor IX, FIX, gene). A major concern with this type of procedure is the potential for a host immune response to the therapeutic gene product, which would render treatment ineffective. Previously, we observed inflammatory, cytotoxic T lymphocyte, and antibody responses to a human FIX (hFIX) transgene product after intramuscular (IM) delivery via an E1/E3-deleted adenoviral vector (Ad-hFIX) in C57BL/6 mice. Different from this Th1-biased immune response, IM injection of adeno-associated viral (AAV) vector, a Th2-biased, non-inflammatory response led to antibody-mediated neutralization of hFIX expression, without CTL activation. In contrast to these observations on muscle-directed vector administration, hepatic AAV-hFIX gene transfer induced immune tolerance to the transgene product (JCI 111:1347). Lack of anti-hFIX formation was demonstrated even after challenge with hFIX in adjuvant. In order to examine the effect of tolerance induction on CD8+ T cell-mediated cellular immune responses, we performed the following experiments. C57BL/6 mice (n=4 per experimental group) received IM injections of AAV-hFIX vector (serotype 1) in one hind limb and/or Ad-hFIX vector in the contra-lateral leg. In the latter case, inflammation (as determined by H&E histological evaluation), CD8+ T cell infiltrate and destruction of hFIX expressing muscle fibers were obvious in both legs because of the Ad-hFIX mediated activation of CTL to hFIX. CD8+ T cell responses were strongest in Ad-hFIX transduced muscle at day 14 and in the AAV-hFIX leg at day 30. Expression of hFIX as determined by immunohistochemistry became undetectable in Ad-hFIX injected muscle by day 30, but was not completely eliminated in AAV-hFIX transduced muscle. Injection of AAV-hFIX only, did not cause inflammation of muscle tissue or CD8+ cell infiltrate. When the identical experiment was carried out in C57BL/6 mice that were expressing hFIX from hepatic gene transfer via the AAV serotype 2 vector (performed 6 weeks earlier), a substantial increase in systemic hFIX expression was observed after IM administration of the Ad and AAV-1 vectors (again injected into contra-lateral legs). However, a portion of the increased expression was subsequently lost, which correlated with inflammation and CD8+ T cell infiltrate of the Ad-hFIX transduced muscle. Interestingly, no (3/4 mice) or only minor (1/4 mice) infiltrate was observed in AAV-hFIX injected muscles. Consequently, hFIX expression persisted in the AAV, but not the Ad transduced legs. Presumably, CTL responses to adenoviral antigens were sufficient to target Ad-hFIX transduced muscle despite tolerance to the transgene product. In contrast to control mice, hepatic tolerized animals failed to form anti-hFIX after challenge by IM injection of these viral vectors. Moreover, inflammatory and destructive cellular immune responses to the transgene product were successfully prevented by hepatic tolerance induction, indicating that tolerance induced by gene transfer to the liver affects cellular as well as antibody-mediated responses and extents to tissues other than liver.