Development of a Liver Gene Therapy Strategy for Haemophilia B With Lentiviral Vectors

Lentiviral vectors (LVs) are attractive tools for liver gene therapy, by virtue of their ability to stably integrate in the genome of target cells and the absence of pre-existing humoral and cellular immunity against vector components in most humans. We have previously reported long-term phenotypic correction of haemophilia B and transgene-specific immune tolerance induction after a single intravenous administration of LVs in mice, provided that transgene expression is stringently targeted to hepatocytes. This is achieved by a combination of transcriptional control, mediated by a synthetic hepatocyte-specific promoter and post-transcriptional control obtained by including in the transgene sequences complementary to the haematopoietic-specific microRNA 142, which binds and targets for degradation any residual transgene mRNA expressed in antigen presenting cells of liver and spleen. We have now evaluated this gene therapy strategy in a large animal model. Our results show long-term canine Factor IX (FIX) expression up to 0.5-1% of normal levels and clinical improvement (almost complete prevention of spontaneous bleedings) in two haemophilia B dogs (>3.5 years cumulative follow up), with mild acute toxicity and without long-term adverse effects nor anti-transgene immune responses. The use of codon-optimised and hyper-functional FIX transgenes increased the potency of LVs (in the pharmacological meaning of efficacy per dose) >15-fold, allowing correction of the disease phenotype at low vector doses in mice, thus improving the therapeutic index of the gene therapy and prompting us to test this improvement in the next treated dog. We have investigated additional improvements in the potency of LVs for liver gene therapy, such as the use of the baculovirus envelope protein gp64, which improves hepatocyte targeting and LV particles resistance to complement-mediated inactivation. We performed a quantitative analysis of LV biodistribution within the liver cell populations and show that Kupffer cells uptake most vector genomes, despite being a small fraction of the total cells, and limit hepatocyte transduction at low administered LV doses. However, pre-treatment with a single dose of a clinically used proteasome inhibitor prior to LV administration reduces this trapping effect and increases hepatocyte transduction and therapeutic efficacy up to 3-fold in haemophilia B mice. We provide evidence that liver gene therapy can establish long-term FIX expression and immune tolerance in mice even in the presence of pre-existing anti-FIX antibody immunity. Since insertional mutagenesis is a concern for integrating vectors, we set out to explore the potential advantages of integrase-defective LVs (IDLVs) to express transgenes in the adult liver, in which hepatocytes turnover is slow. We show that, while not optimal for stable gene replacement therapy in their current design, IDLVs may represent a valuable strategy to induce stable antigen-specific tolerance by transient gene transfer and offer a treatment for immune-mediated diseases. On the other hand, since LV integration is preferable for efficient stable liver gene transfer, we stringently assessed the risk of oncogenesis associated to LV integration in ad hoc mouse models that are sensitised to develop hepatocellular carcinoma and found no detectable increase in carcinogenesis upon liver gene therapy with LVs. Overall our results position LVs as a promising platform for liver gene therapy that may well complement other available vectors to address the different challenges posed by the presentation of haemophilia and its complications in different patients and clinical conditions and may conceivably offer a therapeutic option for lysosomal and metabolic diseases