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4.14
Targeting FVIII Expression to Sinusoidal Cells To Overcome Immunological Responses To Gene Therapy for Hemophilia A

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Targeting FVIII expression to specific cell types to overcome immunological
responses for Hemophilia A gene therapy
Simone Merlin1, Stefania E. Cannizzo1, Valentina Bruscaggin1, Luigi Naldini2 and Antonia Follenzi1.
1 Dept of Health Sciences, Università del Piemonte Orientale, Novara, Italy

2 San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute and Vita Salute San Raffaele University, Milan, Italy

Hemophilia A (HA), a X-linked bleeding disorder, due to mutations in clotting
factor (F) FVIII gene. HA patients are treated with recombinant or
plasma-derived FVIII with high probability to develop inhibitors.
Antigen-presentation occuring in the liver by sinusoidal endothelial cells
and/or Kupffer cells (KC) induces tolerance rather than immunity towards
antigens presented to T-cells. We investigated the role of these cell types in
HA gene transfer using Lentiviral vectors (LV)-expressing FVIII driven by
cell-specific promoters and microRNA target sequences (miRTs).
We prepared LVs containing GFP or BDD-FVIII under the control of the
ubiquitous PGK promoter, the CD11b (monocyte/macrophage-specific) and the VEC
(endothelial-specific) promoter containing several combinations of microRNA
target sequences such as miRT142.3p (silenced in hematopoietic cells) or
miRT126 (silenced in endothelial cells) or miRT122 (silenced in hepatocytes)
and injected HA mice.
After a characterization of the described LV using GFP as transgene expressed
in vivo, we injected HA mice with LV.PGK.FVIII-miRT142, LV.CD11.FVIII-miRT126
and LV.VEC.FVIII-miRT122-142.3p. In the first group anti-FVIII-Abs were
detected 2w after vector delivery, however the presence of miRT142 halved
inhibitors titer in comparison with LV-injected mice without miRT142.3p. Mice
injected with LV.CD11b.FVIII-miRT126 and LV.VEC.FVIII-miRT122-142.3p, reached
long-term phenotypic correction up to 1y with an average of 6-8% FVIII
activity and absence of anti-FVIII-Abs. Moreover, tolerance was obtained
because FVIII was expressed and functional in FVIII-immunized mice producing
FVIII-Abs. In conclusion, LV expressing FVIII under control of cell-specific
promoters combined with miRT-combinations were able to overcome FVIII
off-target expression limiting immune responses and providing phenotypic
correction in treated HA mice.