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Liver gene therapy with adeno-associated viral (AAV) vectors delivering clotting factor transgenes into hepatocytes has shown multiyear therapeutic benefit in adults with hemophilia. However, the mostly episomal nature of AAV vectors challenges their application to young pediatric patients. We developed lentiviral vectors, which integrate in the host cell genome, that achieve efficient liver gene transfer in mice, dogs and non-human primates, by intravenous delivery. Here we first compare engineered coagulation factor VIII transgenes and show that codon-usage optimization improved expression 10-20-fold in hemophilia A mice and that inclusion of an unstructured XTEN peptide, known to increase the half-life of the payload protein, provided an additional >10-fold increase in overall factor VIII output in mice and non-human primates. Stable nearly life-long normal and above-normal factor VIII activity was achieved in hemophilia A mouse models. Overall, we show long-term factor VIII activity and restoration of hemostasis, by lentiviral gene therapy to hemophilia A mice and normal-range factor VIII activity in non-human primate, paving the way for potential clinical application. “Lentiviral gene therapy to the liver establishes stable long-term normal to supra-normal coagulation factor VIII activity in mouse models of hemophilia A and in non-human primates, representing a potential new treatment option for people with hemophilia A.”.

In vivo gene therapy to the liver using lentiviral vectors (LV) may represent a one-and-done therapeutic approach for monogenic diseases. Increasing LV gene therapy potency is crucial for reducing the effective doses, thus alleviating dose-dependent toxicities and facilitating manufacturing. LV-mediated liver transduction may be enhanced by positively selecting LV-transduced hepatocytes after treatment ( a posteriori ) or by augmenting the initial fraction of LV-targeted hepatocytes (a priori). We show here that the a posteriori enhancement increased transgene output without expansion of hepatocytes bearing LV genomic integrations near cancer genes, in mouse models of hemophilia, an inherited coagulation disorder. Furthermore, we enhanced hepatocyte transduction a priori in mice by transiently inhibiting antiviral pathways and/or through a fasting regimen. The most promising transduction-enhancer combination synergized with phagocytosis-shielded LV, resulting in a remarkable 40-fold increase in transgene output. Overall, our work highlights the potential of minimally invasive, cost-effective treatments capable of improving the potency of in vivo LV gene therapy to hepatocytes, in order to expand its applicability and ease clinical translation. Lentiviral vectors are promising gene delivery vehicles to target hepatocytes in vivo, but restriction factors limit their efficiency. Here, the authors counteract many of these restrictions, amplifying lentiviral gene transfer into hepatocytes, strengthening its translational potential.

The efficacy of hemophilia B (HB) replacement therapy is evaluated by coagulation factor IX (FIX) activity in plasma, although FIX bound to extravascular type IV collagen (Col4) also contributes to efficient hemostasis. Here, we investigated the impact of engineering FIX for improved (K5R) or reduced (K5A) Col4 binding on the pharmacokinetic properties of FIX Padua, fused to human serum albumin (HSA QMP ) engineered for favorable neonatal Fc receptor (FcRn) engagement. Hyperactive features and extended plasma half-life in human FcRn expressing mice, attributed to FIX Padua and HSA QMP engineering, respectively, was confirmed. In HB mice, Padua KA -HSA QMP exhibited negligible extravascular distribution and the highest plasma levels at early time points followed by the steepest decay. Conversely, Padua KR -HSA QMP showed increased extravascular distribution and a 3-fold longer functional half-life (80 hours). These findings support the use of Padua KA -HSA QMP and Padua KR -HSA QMP as hyperactive short- or long-term therapeutics, respectively, with opportunities for tailored HB replacement therapy. Coagulation factor IX (FIX) contributes to hemostasis through both plasma activity and binding to extravascular collagen IV. Here, the authors show that collagen binding of albumin-fused hyperactive FIX can be engineered to tailor the pharmacokinetics, distribution, and functional properties in mice for tailored short- or long-term hemophilia B therapy.