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Regulation and Function of Lentiviral Vector-Mediated TCIRG1 Expression in Osteoclasts from Patients with Infantile Malignant Osteopetrosis : Implications for Gene Therapy

Thudium, Christian Schneider LU ; Moscatelli, Ilana LU ; Löfvall, Henrik LU ; Kertész, Zsuzsanna LU ; Montano, Carmen LU ; Bjurström, Carmen Flores LU ; Karsdal, Morten Asser; Schulz, Ansgar; Richter, Johan LU and Henriksen, Kim (2016) In Calcified Tissue International p.1-11
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... (More)

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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author
organization
publishing date
type
Contribution to journal
publication status
in press
subject
keywords
Gene therapy, Lentivirus, Osteoclast, Osteopetrosis, TCIRG1
in
Calcified Tissue International
pages
11 pages
publisher
Springer
external identifiers
  • Scopus:84982311193
ISSN
0171-967X
DOI
10.1007/s00223-016-0187-6
language
English
LU publication?
yes
id
c2f0f206-3ee9-43ff-ab34-aa42df09298e
date added to LUP
2016-09-02 16:06:52
date last changed
2016-09-20 03:03:23
@misc{c2f0f206-3ee9-43ff-ab34-aa42df09298e,
  abstract     = {<p>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<sup>+</sup> cells to IMO CD34<sup>+</sup> 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.</p>},
  author       = {Thudium, Christian Schneider and Moscatelli, Ilana and Löfvall, Henrik and Kertész, Zsuzsanna and Montano, Carmen and Bjurström, Carmen Flores and Karsdal, Morten Asser and Schulz, Ansgar and Richter, Johan and Henriksen, Kim},
  issn         = {0171-967X},
  keyword      = {Gene therapy,Lentivirus,Osteoclast,Osteopetrosis,TCIRG1},
  language     = {eng},
  month        = {08},
  pages        = {1--11},
  publisher    = {ARRAY(0x5446bf0)},
  series       = {Calcified Tissue International},
  title        = {Regulation and Function of Lentiviral Vector-Mediated TCIRG1 Expression in Osteoclasts from Patients with Infantile Malignant Osteopetrosis : Implications for Gene Therapy},
  url          = {http://dx.doi.org/10.1007/s00223-016-0187-6},
  year         = {2016},
}