2003; baxter - Supplements - Haematologica

IV International Workshop on Immune Tolerance in Hemophilia 119Figure 2. Tolerance model. Exposure of neonatal hemophilic animals to sufficiently high levels of human FVIII protein followingFVIII gene transfer may have resulted in the induction of tolerance to human FVIII protein and the FVIII-transduced cells in someof the recipient mice. Conversely, when gene transfer efficiency was low, FVIII expression may have been below a critical thresholdduring the neonatal phase to induce tolerance, resulting in the induction of inhibitory antibodies and/or CTLs directed againstthe FVIII protein and the FVIII-transduced cells, respectively. This tolerance model may account for the heterogeneous immuneresponse in the neonatal hemophilic mice that received FVIII-retroviral vectors. 21 Similarly, prolonged expression of FVIII at highenough levels above a critical threshold may also play a role in establishing post-natal tolerance to the FVIII protein and to theFVIII-transduced cells. 37To further characterize the transduced splenocytesand non-parenchymal liver cells, FACSanalysis was performed on isolated single-cellsuspensions. Most transduced GFP + splenocytesformed typical phagocytic pseudopodia andexpressed high levels of MHC-II, co-stimulatorymolecules CD80 (B7-1) and CD86 (B7-2), celladhesion markers CD106 (VCAM-1), CD31(PECAM-1) and CD54 (ICAM-1) and othermarkers such as CD11b, CD11c and F4/80 characteristicfor antigen-presenting cells (APC).Similarly, these APC-specific markers were alsohighly expressed on some of the transduced GFP +liver cells, possibly Kupffer cells. Splenic andhepatic APCs were transduced in Scid and Balb/cmice, but there were fewer APCs in the GFP +fraction of Balb/c mice compared to Scid, consistentwith an immune rejection of the transducedcells. In addition, efficient transduction ofB lymphocytes was apparent in Balb/c mice,whereas T cells were refractory to lentiviral transduction.Endothelial cell specific markers(CD62E and CD105) were barely expressed onthe GFP + cells, indicating that endothelial cellswere refractory to lentiviral transduction.The relatively efficient lentiviral transductionof APCs should be taken into account whendesigning vectors for gene therapy. Inadvertenttransgene expression in transduced APCs may beundesirable in circumstances where an antibodyresponse against the transgene product shouldbe avoided, 34 particularly in the case of hemophiliagene therapy. It has been shown previouslythat when a ubiquitously expressed promoteris used to express a secretable transgeneproduct, it increases the likelihood of neutralizingantibody formation against this protein.Hence, the use of liver-specific promoters may bewarranted to restrict transgene expression inhepatocytes and to circumvent inadvertent transgeneexpression in APCs following systemiclentiviral vector administration. Follow-up studiesin hemophilic mice and dogs and continuedefforts to improve the vector design are warrant-haematologica vol. 88(supplement n. 12):september 2003