Computationally designed liver-specific transcriptional cis-regulatory modules and hyper-functional factor IX improve liver-targeted gene therapy for hemophilia B.

The development of the next-generation gene therapy vectors for hemophilia requires the use of lower and thus potentially safer vector doses, while augmenting their therapeutic efficacy. We have identified hepatocyte-specific transcriptional cis-regulatory modules (CRMs) using a computational strategy that increased factor IX (FIX) levels 11 to 15-fold. Vector efficacy could be enhanced by combining these hepatocyte-specific CRMs with a synthetic codon-optimized hyper-functional FIX-R338L Padua transgene. This Padua mutation boosted FIX activity up to 7-fold, with no apparent increase in thrombotic risk. We then validated this combination approach using self-complementary serotype 9 adeno-associated viral vectors (scAAV9) in hemophilia B mice. This resulted in sustained supra-physiologic FIX activity (400%), correction of the bleeding diathesis at clinically relevant, low vector doses (5x1010 vg/kg) that are considered safe in subjects undergoing gene therapy. Moreover, immune tolerance could be induced that precluded induction of inhibitory antibodies to FIX upon immunization with recombinant FIX protein.