title: Regulation and Function of Lentiviral Vector-Mediated TCIRG1 Expression in Osteoclasts from Patients with Infantile Malignant Osteopetrosis: Implications for Gene Therapy.
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.
authors.affiliation: Nordic Bioscience, Herlev, Denmark.
authors.forename: Christian Schneider
authors.initials: CS
authors.lastname: Thudium
authors.affiliation: Department of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, BMC A12, 221 84, Lund, Sweden.
authors.forename: Ilana
authors.initials: I
authors.lastname: Moscatelli
authors.affiliation: Nordic Bioscience, Herlev, Denmark.|Department of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, BMC A12, 221 84, Lund, Sweden.
authors.forename: Henrik
authors.initials: H
authors.lastname: Löfvall
authors.affiliation: Department of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, BMC A12, 221 84, Lund, Sweden.
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Kertész
authors.affiliation: Department of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, BMC A12, 221 84, Lund, Sweden.
authors.forename: Carmen
authors.initials: C
authors.lastname: Montano
authors.affiliation: Department of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, BMC A12, 221 84, Lund, Sweden.
authors.forename: Carmen Flores
authors.initials: CF
authors.lastname: Bjurström
authors.affiliation: Nordic Bioscience, Herlev, Denmark.
authors.forename: Morten Asser
authors.initials: MA
authors.lastname: Karsdal
authors.affiliation: Department of Pediatrics and Adolescent Medicine, University Medical Center, Ulm, Germany.
authors.forename: Ansgar
authors.initials: A
authors.lastname: Schulz
authors.affiliation: Department of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, BMC A12, 221 84, Lund, Sweden. Johan.Richter@med.lu.se.
authors.forename: Johan
authors.initials: J
authors.lastname: Richter
authors.affiliation: Nordic Bioscience, Herlev, Denmark.
authors.forename: Kim
authors.initials: K
authors.lastname: Henriksen
mesh_terms: D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus; D010010:Osteoclasts / Q000378:metabolism*; D010022:Osteopetrosis / Q000235:genetics*; D025262:Vacuolar Proton-Translocating ATPases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: C472421:TCIRG1 protein, human; D025262:Vacuolar Proton-Translocating ATPases
keywords: Gene therapy; Lentivirus; Osteoclast; Osteopetrosis; TCIRG1