Adenoviral Mediated TGF-β1inhibition in a Murine Model of Myelofibrosis Can Prevent and Cure Bone Marrow Fibrosis Development.

Myelofibrosis is a clinical feature of several hematopoietic disorders and is most prominent in idiopathic myelofibrosis. It is characterized by excessive deposits of extracellular matrix proteins which occur as a marrow microenvironment reactive response to cytokines released from the clonal malignant myeloproliferation. The observation that mice exposed to high systemic levels of thrombopoietin (TPO) invariably develop a myelofibrosis within a period of 4 to 8 weeks has allowed the demonstration of the crucial role of TGF-b1 released by hematopoietic cells in the promotion of myelofibrosis (Chagraoui et al Blood, 2002 ; 100 :3495). The aim of this study was to investigate whether TGF-b1 inhibition could directly inhibit the fibrosis development in a curative approach of this murine model.

An adenovirus encoding for TGF-b1 soluble receptor was constructed. SCID mice were infected with this adenovirus to measure the TGF-b1 inhibitory effect. We measure TGF-b1-Rs (TGF-b1 soluble receptor) concentration and TGF-b1 levels in 5 groups of mice injected with 0- 6.5 106- 6.5 107- 6.5 108-and 6.5 109 pfu/mice. We demonstrated that 6.5 108 pfu/mice was able to completely inhibit 10 fold increase of circulating TGF-b1 and that 1 microgram of soluble receptor was able to inhibit 1 ng of TGF-b1.

All subsequent experiments were performed by injecting 1 109 pfu/mice either shortly after transplantation (preventive) or after 30 days (curative). Briefly, 46 mice (2 individuals experiments n= 16 and n= 30) were transplanted with 5-FU treated syngeneic bone marrow cells transduced with a retrovirus encoding for murine TPO. Retroviral transduction efficiency varied from 67% to 87%. All mice developed a myeloproliferative syndrome characterized by an increase in platelet count, a leukocytosis, an increase in progenitor cells in the blood circulation and a 1000-fold increase in TPO level. TGF-b Rs (13 to 32 micrograms/ml) was detected in the blood of all mice injected with the TGF-b1-Rs Adeno. A dramatic decrease in TGF-b1 level was noticed in all these treated mice in comparison to the control groups. Histological analysis demonstrated that the two approaches (curative or preventive) were identical to abolish fibrosis development in bone marrow as well as in the spleen. However, all mice which received the TGF-b1-RS adenovirus developed a hepatitis leading to death 6 weeks to 3 months after infections.

In conclusion our approach support the major role of TGF-b1 in myelofibrosis development as previously demonstrated by our group using TGF-b1 KO mice. Moreover, the present results suggest that TGF-b1 inhibition could be a relevant approach to treat patients presenting a TGF-b1 mediated bone marrow fibrosis.