Abstract
Infantile malignant osteopetrosis (IMO) is a rare, recessive disorder characterized by increased bone mass caused by dysfunctional osteoclasts. The disease is most often caused by mutations in the TCIRG1 gene encoding a subunit of the V-ATPase involved in the osteoclasts capacity to resorb bone. We previously showed that osteoclast function can be restored by lentiviral vector-mediated expression of TCIRG1, but the exact threshold for restoration of resorption as well as the cellular response to vector-mediated TCIRG1 expression is unknown. Here we show that expression of TCIRG1 protein from a bicistronic TCIRG1/GFP lentiviral vector was only observed in mature osteoclasts, and not in their precursors or macrophages, in contrast to GFP expression, which was observed under all conditions. Thus, vector-mediated TCIRG1 expression appears to be post-transcriptionally regulated, preventing overexpression and/or ectopic expression and ensuring protein expression similar to that of wild-type osteoclasts. Codon optimization of TCIRG1 led to increased expression of mRNA but lower levels of protein and functional rescue. When assessing the functional rescue threshold in vitro, addition of 30 % CB CD34+ cells to IMO CD34+ patient cells was sufficient to completely normalize resorptive function after osteoclast differentiation. From both an efficacy and a safety perspective, these findings will clearly be of benefit during further development of gene therapy for osteopetrosis.
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Acknowledgments
We thank Christopher Baum and Axel Schambach (Hannover Medical School, Germany) for providing us with the lentiviral vector backbone. KH is supported by the Danish Research Foundation (Den Danske Forskningsfond). JR is supported by grants from the Swedish Childhood Cancer Foundation, a Clinical Research Award, from Lund University Hospital, the Foundations of Lund University Hospital. AS is partially supported by grants of the EU (ERARE initiative, project OSTEOPETR). HL is supported by Marie Curie Initial Training Networks (Euroclast, FP7-People-2013-ITN: # 607446). The Lund Stem Cell Center is supported by a Center of Excellence grant in life sciences from the Swedish Foundation for Strategic Research. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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Morten Asser Karsdal owns stock in Nordic Bioscience. Christian Schneider Thudium, Ilana Moscatelli, Henrik Löfvall, Zsuzsanna Kertész, Carmen Montano, Carmen Flores Bjurström, Ansgar Schulz, Johan Richter and Kim Henriksen declare no competing financial interests.
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Thudium, C.S., Moscatelli, I., Löfvall, H. et al. Regulation and Function of Lentiviral Vector-Mediated TCIRG1 Expression in Osteoclasts from Patients with Infantile Malignant Osteopetrosis: Implications for Gene Therapy. Calcif Tissue Int 99, 638–648 (2016). https://doi.org/10.1007/s00223-016-0187-6
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DOI: https://doi.org/10.1007/s00223-016-0187-6