Page 130 - 2021_06-Haematologica-web

R. Famà et al.
with Lyve-1 was still visible 1 month post-injection, with a few F4/80+GFP+ cells appearing in the pF8-1175 and pF8- 600 injected mice (Online Supplementary Figure S4A and B).
As previously observed for pF8-117523 in the spleen at 2 weeks, all pF8 short forms resulted in very low co-staining with the putative endothelial marker CD31 (Figure 6, left panel), while pF8-1,175, pF8-600 and pF8-446 showed a partial GFP expression in F4/80+ cells (Figure 6, right panel). Interestingly, in these mice several GFP+ cells were evident around the germinal centers (GC), resembling the EC lining in the marginal sinus of the spleen GC.35 Unlike other pro- moters, the spleens of mice receiving the LV.pF8-342, showed a higher percentage of F4/80+/GFP+ cells (Figure 6, right panel) 2 weeks after the LV injection. The same GFP distribution pattern, but with a reduced expression, was confirmed in all treated mice at 1 and 2 months post LV- delivery. In particular, the LV.pF8-342.GFP-injected mice showed the most relevant reduction of GFP expression in F4/80+ cells over time (Online Supplementary Figures S5 and S6). Taken together, these results demonstrate that a signif- icant reduction in pF8 size does not compromise its activi- ty or specificity, suggesting that pF8-342 is enough for maintaining an endothelial-specific expression.
In vivo long-term recovery of factor VIII activity in a hemophilia A mouse model using the shortened F8 promotor
One of the major obstacles in HA gene therapy is repre- sented by the large size of the FVIII expression cassette. As such, the choice of a suitable promoter to drive its expres- sion is fundamental for success. In order to explore the ability of our new shortened promoters in rescuing FVIII expression in vivo, we generated LV carrying FVIII under their control and injected 1x109 TU in B6/129 HA mice (n=4-6). pF8-1175 was used as a control. The FVIII activity promoted by pF8-600, pF8-446 resulted in a long-term (28 weeks) therapeutic correction (~10%), with no differences seen among them, and with respect to pF8-1175 (Figure 7A). Unexpectedly, despite the reduced response to Ets showed by luciferase assays, also the pF8-342 resulted in long-term therapeutic correction comparable to the other promoters tested. Importantly, independent of the size of the promoter used in the transfer construct, no anti-FVIII antibodies were detected in all treated mice, indicating the maintenance of specificity and, then, of an immunological profile similar to those observed for pF8-117523 (Figure 7B). High levels of anti-FVIII antibodies were instead detected
A
B
Figure 7. In vivo long-term correction of factor VIII activity with no immune response by F8 shortened promoters. (A) Graphic representation of factor VIII (FVIII) activ- ity of B6/129 HA mice injected with lentiviral vecor (LV) LV.pF8-600.FVIII (grey square, n=6), LV.pF8-446.FVIII (light blue rhombus, n=4) or LV.pF8-342.FVIII (green tri- angle, n=4). pF8-1175.FVIII (black circle, n=4) were used as a control, while untreated hemophilia A (HA) mice were used as the negative control (violet X, n=3). No statistical differences in FVIII activity levels were observed among the four LV-injected experimental groups. (B) Enzyme-linked immunosorbent assay showing the absence of anti-FVIII antibodies in plasma of HA mice after LV delivery. Anti-FVIII positive HA mice injected with LV.PGK-FVIII served as positive control, while untreated HA mice were used as the negative control. Plasma dilution 1:2,000. Color bars were maintained according to the color scale used for FVIII activity graph.
1632
haematologica | 2021; 106(6)