Hematopoietic Stem Cells as Targets for Gene Therapy of Hemophilia A

Considering the plasticity of hematopoietic stem cells (HSC), they would be ideal targets for gene therapy of hemophilia A by virtue of their progeny providing immediate access to the blood stream. However, several attempts to show expression of recombinant factor VIII (rFVIII) by primary hematopoietic cells and cell lines have failed, which was attributed to the inability of HSC to secrete rFVIII. Here we describe the generation of stable, FVIII-secreting hematopoietic cell lines representing different blood-cell types using a bicistronic lentiviral vector encoding for a B-domain deleted FVIII (FVIII△B) and enhanced green fluorescence protein (EGFP). Transduced cell lines with erythroid and/or megakaryocytic background, (K562-F8 and TF-1-F8), secrete high levels of FVIII in the order of 76.4 and 41.6 ng FVIII:C/ml, while moderate and low levels are observed in B-lymphoblastoid Raji-F8 cells and the T leukemia line Jurkat-F8 which secrete 6.73 and 1.83 ng FVIII:C/ml, respectively. The capacity to secrete rFVIII appeared to depend on factors related to the cell lineage, rather than on the transduction efficacy. The established cell lines should be helpful in further elucidating mechanisms which are able to improve FVIII secretion in hematopoietic cells on a post-translational level and suggest reanalysis of hematopoietic cells as target for gene therapy of the hemophiliacs.