Platelet-targeted hyperfunctional FIX gene therapy for hemophilia B mice even with preexisting anti-FIX immunity

Abstract

Gene therapy may lead to a cure for hemophilia B (HB) if it is successful. Data from clinical trials using adeno-associated virus (AAV)–mediated liver-targeted FIX gene therapy are very encouraging. However, this protocol can be applied only to adults who do not have liver disease or anti-AAV antibodies, which occur in 30% to 50% of individuals. Thus, developing a protocol that can be applied to all HB patients is desired. Our previous studies have demonstrated that lentivirus-mediated platelet-specific FIX (2bF9) gene therapy can rescue bleeding diathesis and induce immune tolerance in FIXnull mice, but FIX expression was only ∼2% to 3% in whole blood. To improve the efficacy, we used a codon-optimized hyperfunctional FIX-Padua (2bCoF9R338L) to replace the 2bF9 cassette, resulting in 70% to 122% (35.08-60.77 mU/108 platelets) activity levels in 2bCoF9R338L-transduced FIXnull mice. Importantly, sustained hyperfunctional platelet-FIX expression was achieved in all 2bCoF9R338L-transduced highly immunized recipients with activity levels of 18.00 ± 9.11 and 9.36 ± 12.23 mU/108 platelets in the groups treated with 11 Gy and 6.6 Gy, respectively. The anti-FIX antibody titers declined with time, and immune tolerance was established after 2bCoF9R338L gene therapy. We found that incorporating the proteasome inhibitor bortezomib into preconditioning can help eliminate anti-FIX antibodies. The bleeding phenotype in 2bCoF9R338L-transduced recipients was completely rescued in a tail bleeding test and a needle-induced knee joint injury model once inhibitors dropped to undetectable. The hemostatic efficacy in 2bCoF9R338L-transduced recipients was further confirmed by ROTEM and thrombin generation assay (TGA). Together, our studies suggest that 2bCoF9R338L gene therapy can be a promising protocol for all HB patients, including patients with inhibitors.

Targeting transmembrane-domain-less MOG expression to platelets prevents disease development in experimental autoimmune encephalomyelitis

“…As shown in Figure 4G, MOG TD and MOG FL proviral DNA were detected by PCR in the 2bMOG TDand 2bMOG FL -transduced recipients, respectively. The copy number of proviral DNA in the 2bMOG TD and 2bMOG FL groups were comparable (Figure 4H) when determined by qPCR using primers to amplify the LTR sequence as previously reported (30). These data suggest that the failure of platelet-targeted MOG TD expression in ameliorating EAE is not due to a loss of proviral DNA insertion.…”

“…There was no detectable MOG protein expression in the platelets from 2bMOG TD -transduced recipients, regardless of surface- or intracellular-staining ( Figures 4E, F ), and platelet-targeted MOG TD expression did not affect the development of EAE ( Figures 4A–D ) in mice. To examine cell viability in 2bMOG TD -transduced recipients, we used semi-quantitative PCR and quantitative real-time PCR (qPCR) ( 30 ) to quantify the MOG proviral DNA. As shown in Figure 4G , MOG TD and MOG FL proviral DNA were detected by PCR in the 2bMOG TD - and 2bMOG FL -transduced recipients, respectively.…”

“…Our previous studies have shown that platelet targeted gene transfer can effectively induce antigen-specific immune tolerance in hemophilia A and B mice even with pre-existing immunity (24,(26)(27)(28)(29)(30)46). The present study provides proof-of concept that platelet-targeted gene therapy approach could be applied to induce immune tolerance in autoimmune disease model EAE.…”

“…Our previous studies have demonstrated that targeting coagulation factor VIII (FVIII) expression to platelets under the control of the plateletspecific aIIb promoter (2bF8) results in the storage of FVIII in platelet a-granules and that platelet-derived FVIII can effectively induce antigen-specific immune tolerance in hemophilia A mice even with pre-existing anti-FVIII immunity (23)(24)(25)(26)(27)(28). When a similar approach was applied to a hemophilia B model, which results from factor FIX (FIX) deficiency, antigen-specific immune tolerance was induced in hemophilia B mice even with anti-FIX immunity (29,30). Furthermore, targeting noncoagulant protein ovalbumin (OVA) to platelets under the same promoter used for the FVIII and FIX studies also resulted in neoprotein OVA storage in platelet a-granules, leading to antigen-specific immune tolerance (31,32).…”

Gene Therapy Approaches for the Treatment of Hemophilia B

“…It has been shown that, despite the presence of exogenous factor IX in this approach, platelet-specific therapy does not cause anaphylaxis and the formation of anti-FIX antibodies but, on the contrary, leads to the elimination of preexisting antibodies and can provide the production of factor IX after their elimination. Notably, the combination of a highly effective F9 -Padua variant, irradiation, and a proteasome inhibitor accelerated the elimination of antibodies [ 59 ].…”