The development of the next-generation gene therapy vectors for hemophilia requires using lower and thus potentially safer vector doses and augmenting their therapeutic efficacy. We have identified hepatocyte-specific transcriptional cis-regulatory modules (CRMs) by 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 hyperfunctional FIX-R338L Padua transgene. This Padua mutation boosted FIX activity up to sevenfold, with no apparent increase in thrombotic risk. We then validated this combination approach using self-complementary adenoassociated virus serotype 9 (scAAV9) vectors in hemophilia B mice. This resulted in sustained supraphysiologic FIX activity (400%), correction of the bleeding diathesis at clinically relevant, low vector doses (5 × 10(10) vector genomes [vg]/kg) that are considered safe in patients undergoing gene therapy. Moreover, immune tolerance could be induced that precluded induction of inhibitory antibodies to FIX upon immunization with recombinant FIX protein.