Ex Vivo Gene Therapy Approach By Targt Technology for the Treatment of β-Thalassemia Intermedia

β-Thalassemia intermedia or non-transfusion dependent thalassemia (NTDT) is a genetic blood disorder characterized by ineffective erythropoiesis (IE), anemia, splenomegaly and systemic iron overload mediated by down-regulation of hepcidin. Using Hbb^th3/+animals affected by NTDT, we have shown that iron restriction improves anemia and reduces splenomegaly by reducing the formation of hemichromes and improving the lifespan of red blood cells (RBC; Gardenghi et al, JCI, 2010; Guo et al, JCI, 2013; Casu et all Blood 2016). These findings emphasize the strong correlation between erythropoiesis, RBC synthesis and iron metabolism. In theory, agents that can stimulate erythropoiesis (such as erythropoietin or EPO) could improve anemia by increasing the production of RBC and, under conditions of reduced iron absorption, correct the iron overload by consuming the excess of iron stored in organs. Here we used a new ex vivo technology called TARGT (Transduced Autologous Restorative Gene Therapy) for prolonged production and secretion of therapeutic agents. Primary dermal fibroblasts transduced with helper dependent adenovirus expressing mouse Epo were embedded in Matrigel and implanted in the dorsal area of 2-3 months old Hbb^th3/+and wild-type (WT) C57BL/6 mice. Three groups were generated for each genotype using different amounts of genetically modified fibroblasts (10e5, 5x10e5 and 1x10e6 transduced cells), while one group received no transduced fibroblasts as control. Complete blood count and serum harvest for Epo levels measurement were performed one week after implantation and then every ten days until the end of the experiment, at six weeks. In WT mice, already one week after fibroblasts implantation, hemoglobin (Hb) levels raised 2/3 g/dL, showing erythrocytosis. At the end of the treatment animals showed significantly increased Hb levels (18.5±1.7 g/dL, 19.4±0.5 g/dL and 19.8±0.8 g/dL in the groups implanted with 10e5, 5x10e5 and 1x10e6 fibroblasts, respectively, compared to 13.5±0.8 g/dL in controls). RBC numbers and HTC follow the same trend, showing increases in the range of 27% to 39%.

In Hbb^th3/+mice Hb levels rose about 3 g/dl already one week after fibroblasts implantation and remained elevated until the end experiment (8.7±0.3 g/dL, 10.7±1.0 g/dL and 10.6±1.8 g/dL in the groups implanted with 10e5, 5x10e5 and 1x10e6 fibroblasts, respectively, compared to 7.7±0.7 g/dL in controls). RBC numbers and HTC follow the same trend, showing increases in the range of 7% to 27%. At the end of the treatment, serum Epo levels were markedly increased at all doses both in Hbb^th3/+and WT animals (more than 50% and up to 90%) when compared with their respective controls.

However, as expected, stimulation of erythropoiesis led to worsening of splenomegaly and suppression of hepcidin, likely preventing the beneficial effect of erythroid-mediated consumption of stored iron in Hbb^th3/+animals. Here we postulated that, in presence of agents that increase erythropoiesis such as Epo, some level of iron restriction is required to improve the anemia, prevent exacerbation of the splenomegaly and, concurrently, decreased iron overload. As a proof of principle of the ability of this technology to improve anemia and decrease iron overload, TARGTEPO was combined with low iron diet or iron chelation. We expect that decreased iron intake will improve the quality of the RBC, while the larger number of RBC produced should utilize more stored iron, reversing the iron overload in Hbb^th3/+animals. Preliminary results at three weeks from the beginning of the experiment indicated that in WT implanted-animals the combination of TARGTEPO with low iron diet significantly reduced Hb levels (-40%), HTC (-42%), RBC number (-38%) and reticulocytes (-80%) when compared to animals overexpressing Epo and receiving normal iron diet or iron chelation. This indicates that in WT animals, after 3 weeks, the stored iron is insufficient to support the increased erythropoiesis. In contrast, in Hbb^th3/+animals this drop in Hb was not seen, suggesting that this effect is delayed due to the highest level of stored iron. Complete characterization of these models and their parameters is in progress. These preliminary results suggest that the TARGTplatformcould be utilized, in combination with drugs that limit iron intake, to improve anemia and decrease iron overload in NTDT. Additional experiments are in progress to test this hypothesis.