Effects of FVIII immunity on hepatocyte and hematopoietic stem cell–directed gene therapy of murine hemophilia A

Abstract

Immune responses to coagulation factors VIII (FVIII) and IX (FIX) represent primary obstacles to hemophilia treatment. Previously, we showed that hematopoietic stem cell (HSC) retroviral gene therapy induces immune nonresponsiveness to FVIII in both naive and preimmunized murine hemophilia A settings. Liver-directed adeno-associated viral (AAV)-FIX vector gene transfer achieved similar results in preclinical hemophilia B models. However, as clinical immune responses to FVIII and FIX differ, we investigated the ability of liver-directed AAV-FVIII gene therapy to affect FVIII immunity in hemophilia A mice. Both FVIII naive and preimmunized mice were administered recombinant AAV8 encoding a liver-directed bioengineered FVIII expression cassette. Naive animals receiving high or mid-doses subsequently achieved near normal FVIII activity levels. However, challenge with adjuvant-free recombinant FVIII induced loss of FVIII activity and anti-FVIII antibodies in mid-dose, but not high-dose AAV or HSC lentiviral (LV) vector gene therapy cohorts. Furthermore, unlike what was shown previously for FIX gene transfer, AAV-FVIII administration to hemophilia A inhibitor mice conferred no effect on anti-FVIII antibody or inhibitory titers. These data suggest that functional differences exist in the immune modulation achieved to FVIII or FIX in hemophilia mice by gene therapy approaches incorporating liver-directed AAV vectors or HSC-directed LV.

Complexity of immune responses to AAV transgene products – Example of factor IX

“…In contrast to the canine model, reversal in hemophilia A mice by hepatic AAV gene transfer has been challenging [95]. This may reflect differences in generation of long-lived plasma cells between the two species in response to FVIII, which may be harder to suppress.…”

“…This may reflect differences in generation of long-lived plasma cells between the two species in response to FVIII, which may be harder to suppress. However, preventive tolerance induction has been accomplished in the mice using optimized expression cassettes [84, 95, 96]. High levels of FVIII expression in hepatocytes has some potential for induction of a cellular stress response, which however does not appear to increase immunogenicity [96, 97].…”

Adenovirus vectors in hematopoietic stem cell genome editing

“…The results are undoubtedly encouraging. Instead of liver-directed gene transfer, HSC-directed F8 gene transfer has been investigated and showed advantages in tackling pre-existing immunity [237]. Hence, the search for alternative approaches could address these limitations and expand our treatment options.…”

“…Efficient targeted integration of a F9 gene was also demonstrated by using a single HDAd vector simultaneously carrying CRISPR/Cas9 and a donor cassette [105]. Instead of liver-directed gene transfer, HSC-directed F8 gene transfer has been investigated and showed advantages in tackling pre-existing immunity [237]. We recently reported on an approach for HSCderived, erythroid-specific F8 production based on HDAd5/35++ vectors [151].…”

Protein S: A multifunctional anticoagulant

“…Drawbacks to the current hemophilia treatments have prompted researchers to seek alternatives, such as discovery of longer-acting factors that can be used at lower doses, utilization of non-factor therapies, and gene therapy. Gene therapy particularly is expected to be the key to curing hemophilia [34, 35]. …”