Regulation and Function of Lentiviral Vector-Mediated TCIRG1 Expression in Osteoclasts from Patients with Infantile Malignant Osteopetrosis: Implications for Gene Therapy
| Autor: | Kim Henriksen, Johan Richter, Carmen Montano, Christian S. Thudium, Ansgar Schulz, Zsuzsanna Kertész, Henrik Löfvall, Ilana Moscatelli, Morten A. Karsdal, Carmen Flores Bjurström |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Vacuolar Proton-Translocating ATPases Endocrinology Diabetes and Metabolism Genetic enhancement Genetic Vectors Osteoclasts 030209 endocrinology & metabolism Biology Viral vector Green fluorescent protein TCIRG1 03 medical and health sciences 0302 clinical medicine Endocrinology Osteoclast medicine Humans Orthopedics and Sports Medicine Lentivirus Osteopetrosis Genetic Therapy medicine.disease 030104 developmental biology medicine.anatomical_structure Cancer research Ectopic expression Infantile malignant osteopetrosis |
| Zdroj: | Calcified tissue international. 99(6) |
| ISSN: | 1432-0827 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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