Haemophilia B might be permanently cured by gene therapy--the introduction of a correct copy of the factor IX gene into the somatic cells of a patient. Here, we have introduced a recombinant human factor IX cDNA into primary human keratinocytes by means of a defective retroviral vector. In tissue culture, transduced keratinocytes were found to secrete biologically active factor IX and after transplantation of these cells into nude mice, human factor IX was detected in the bloodstream in small quantities for one week. This is the first demonstration of a therapeutic protein reaching the bloodstream from transduced primary keratinocytes. This may have implications for the treatment of haemophilia B and other disorders.

Haemophilia B patients are normally treated, either prophylactically or in response to bleeding episodes, by frequent intravenous injections of factor IX purified from blood donors. Here we show in model animal experiments that purified human factor IX, when injected subcutaneously, is rapidly (in 3-11 h) and reasonably efficiently (30-40% of an equivalent intravenous dose) transported at least partly by the lymphatic drainage of the skin into the bloodstream, mostly in a biologically active form. This suggests that patients could be treated prophylactically by subcutaneous rather than intravenous injection, where the short delay in raising plasma factor IX to haemostatic levels would be clinically acceptable. More generally, our studies emphasize that the subcutaneous route of injection should be useful for other therapeutic proteins, including other clotting factors, which have to be delivered to the bloodstream, as long as their half-life is at least a few hours allowing time for transport into the general circulation.

We have been investigating the use of human keratinocytes as potential target cells for gene therapy for haemophilia B, with the aim of curing haemophilia by means of a factor IX secreting skin graft. Previous studies indicated that keratinocytes might be suitable cells, although a potential problem was that the recombinant factor IX secreted by transduced keratinocytes was found to be only 40% biologically active. We now report, using an alternative assay to test for activity, that the secreted factor IX appears to be almost fully active.

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