Bone Abstracts

Infantile malignant osteopetrosis (IMO) is a rare, lethal, recessive disorder characterized by dysfunctional osteoclasts. TCIRG1, encoding the osteoclast V-ATPase, is mutated in 50% of IMO patients. We have previously shown that the resorptive function in osteoclasts derived from IMO patients can be restored in vitro by expressing TCIRG1 using a lentiviral vector. In this study, we aim to investigate the cellular response to vector-derived TCIRG1 expression and to determine the TCIRG1 expression needed to restore resorptive function.

CD34⁺ cells from peripheral blood of IMO patients were transduced with a lentiviral vector expressing TCIRG1 and GFP under the SFFV promoter. The cells were expanded for 2 weeks followed by in vitro differentiation towards osteoclasts or macrophages for 3, 5, 9 or 14 days. TCIRG1 expression was analyzed by western blot and was only observed in mature osteoclasts in contrast to GFP which was observed under all conditions.

The threshold needed to restore resorptive function in vitro was assessed by mixing CD34⁺ cord blood (CB) cells or transduced CD34⁺ IMO cells with untreated CD34⁺ IMO cells and differentiating them into osteoclasts on bone slices. TRAP activity and CTX-I release were measured in the media. Mixing 30% CB cells with IMO cells was sufficient to completely normalize resorptive function measured by resorption per osteoclast (CTX-I/TRAP). Doses as low as 2.5% transduced IMO cells mixed with untreated IMO cells were capable of increasing the resorption per osteoclast reaching up to a 14-fold increase at 30% transduced IMO cells.

In conclusion we show that lentiviral-mediated expression of TCIRG1 is regulated in the same manner as endogenous TCIRG1 despite being expressed by a constitutively active promoter. This suggests that TCIRG1 is post-transcriptionally regulated. We furthermore show that only a low fraction of human pre-osteoclasts with functional TCIRG1 is needed to significantly increase resorptive function in vitro.