Non-Genotoxic Conditioning to Increase Gene Therapy Safety in a Rare Bone Disease

Autosomal recessive osteopetrosis (ARO) is a rare genetic disease, affecting osteoclast differentiation or function. Most ARO patients present mutations in TCIRG1 gene, encoding the a3 subunit of V-ATPase proton pump, necessary for the acidification of bone resorption lacunae by osteoclasts. Symptoms include dense and brittle bones, limited bone marrow cavity, anaemia and progressive nerve compression, leading to death in the first decade of life. To date, hematopoietic stem cell transplantation (HSCT) is the only therapeutic option, but it is limited by availability of HLA-matched donors, toxicity of conditioning regimens and significant morbidity. We and others proposed gene therapy (GT) as an alternative strategy to overcome donor-related issues. However, the burden of conventional myeloablative conditioning on patients remains a strong unmet clinical need. Thus, we evaluated the use of a novel non-genotoxic conditioning in GT setting of TCIRG1-dependent ARO. In particular, we tested antibody-drug conjugates (ADCs), formed by anti-cKit or anti-CD45 antibody conjugated to the saporin toxin, able to make space in bone marrow (BM) and to promote hematopoietic stem and progenitor cell (HSPC) engraftment without off-target toxicity in adult mice. Since ARO symptoms occur very early in life, we evaluated the HSPC depleting potential of ADCs on WT newborn mice, administered via temporal vein at 1 day of life. We sacrificed mice 2 days after treatment and observed partial HSPC depletion in BM, spleen and peripheral blood. Notably, we did not observe histological lesions or increase in apoptosis (evaluated with Caspase 3 immunohistochemistry) due to organ toxicity in kidney, brain and BM of ADC-treated mice. To evaluate the efficacy of non-genotoxic conditioning in favouring HSPC engraftment early in life, we performed mismatched HSCT in WT ADC-conditioned mice, and compared it to transplantation after sublethal total body irradiation or without conditioning. Twenty weeks after transplant, we observed low but persistent donor chimerism in ADC-treated mice compared to irradiated controls in peripheral blood, BM, spleen and thymus.As reported in literature, very low level of donor cells engraftment is sufficient to restore bone phenotype in osteopetrotic mice. We hypothesized that ADC conditioning could successfully guarantee bone phenotype amelioration and reduction of the conditioning toxicity in the osteopetrotic mouse model. This approach may be even more advantageous in the GT setting, in which autologous HSCT avoids the risk of graft rejection. We plan to apply ADC conditioning on osteopetrotic mice before the transplant of lentiviral vector GT Lin- cells, to test the efficacy of our strategy on this severe bone disease model. Acknowledgements. This project has received funding from the European Calcified Tissue Society