Bone 57 (2013) 1–9

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Original Full Length Article

Lentiviral gene transfer of TCIRG1 into peripheral blood CD34+ cellsrestores osteoclast function in infantile malignant osteopetrosis☆

Ilana Moscatelli a,1, Christian Schneider Thudium a,b,1, Carmen Flores a, Ansgar Schulz c, Maria Askmyr a,Natasja Stæhr Gudmann b, Nanna Merete Andersen b, Oscar Porras d, Morten Asser Karsdal b, Anna Villa e,f,Anders Fasth g, Kim Henriksen b, Johan Richter a,⁎a Department of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, Lund, Swedenb Nordic Bioscience, Denmarkc Department of Pediatrics and Adolescent Medicine, University Medical Center Ulm, Germanyd Department of Immunology, National Children's Hospital, San Jose, Costa Ricae Milan Unit, Istituto di Ricerca e Genetica Biomedica, CNR, Milan, Italyf Istituto Clinico Humanitas, Rozzano, Milano, Italyg Department of Pediatrics, University of Gothenburg, Gothenburg, Sweden

☆ The work was performed in Lund, Sweden and Copen⁎ Corresponding author at: Department ofMolecularM

Strategic Center for Stem Cell Biology, Lund 221 84, SwedE-mail address: johan.richter@med.lu.se (J. Richter).

1 These authors contributed equally to this work.

8756-3282/$ – see front matter © 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.bone.2013.07.026

a b s t r a c t

a r t i c l e i n f o

Article history:Received 25 March 2013Revised 12 June 2013Accepted 17 July 2013Available online 29 July 2013

Edited by: R. Baron

Keywords:Infantile malignant osteopetrosisTCIRG1Lentiviral gene transferHematopoietic stem cellsOsteoclasts

Infantile malignant osteopetrosis (IMO) is a rare, lethal, autosomal recessive disorder characterized by non-functional osteoclasts. More than 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunitof the osteoclast proton pump. The aim of this study was to restore the resorptive function of IMO osteoclasts bylentiviralmediated gene transfer of the TCIRG1 cDNA. CD34+ cells fromperipheral blood of five IMOpatients andfrom normal cord blood were transduced with lentiviral vectors expressing TCIRG1 and GFP under a SFFV pro-moter, expanded in culture and differentiated on bone slices to mature osteoclasts. qPCR analysis and westernblot revealed increased mRNA and protein levels of TCIRG1, comparable to controls. Vector corrected IMO oste-oclasts generated increased release of Ca2+ and bone degradation product CTX-I into the media as well as in-creased formation of resorption pits in the bone slices, while non-corrected IMO osteoclasts failed to resorbbone. Resorption was approximately 70–80% of that of osteoclasts generated from cord blood. Furthermore,transduced CD34+ cells successfully engrafted in NSG-mice. In conclusion we provide the first evidence oflentiviral-mediated correction of a human genetic disease affecting the osteoclastic lineage.

© 2013 Elsevier Inc. All rights reserved.

Introduction

Osteoclasts are derived from hematopoietic stem cells (HSC) viathe monocytic lineage, and in the presence of the osteoblast derivedcytokines M-CSF and RANKL differentiate into multinucleated boneresorbing cells [1]. The ability of the osteoclasts to dissolve the calcifiedbonematrix is key to the remodeling process and hencemaintenance ofthe skeleton. Dissolution of the bone matrix requires a combination ofacidification by hydrochloric acid and secretion of proteases into the re-sorption lacunae [1]. Acidification is mediated by the active transport ofprotons into the resorption lacunae by the V-ATPase, and the secretionof chloride by the chloride anti-porter ClC-7 [2–4].

Osteopetrosis comprises a heterogeneous group of heritable condi-tions characterized by the lack of osteoclast mediated bone resorption

hagen, Denmark.edicine and Gene Therapy, Lunden. Fax: +46 46 222 05 68.

ghts reserved.

and abnormal bone development [5]. Infantile malignant osteopetrosis(IMO) is the most severe form of osteopetrosis. The incidence of IMO isapproximately 1 in 300,000but is almost 10 times higher in Costa Rica [6].

More than 50% of the patients have mutations in the TCIRG1 geneencoding the a3 subunit of the V-ATPase proton pump, which is neces-sary for the acidification of the resorption lacuna. Childrenwith this dis-order have a normal or elevated number of non-resorbing osteoclasts[2,3,7,8], which leads to severely hampered bone remodeling, resultingin dense and brittle bones [9]. This in turn causes bone marrow failurefollowed by anemia and hepatosplenomegaly, furthermore, compres-sion of nervus opticus leads to blindness [10,11].

HSC transplantation is presently the only curative form of treatmentfor IMO, but is associated with high mortality when an HLA-identicaldonor is not available [12]. Thus development of alternative treatmentssuch as gene therapy is highly warranted [13]. We have previouslyshown that the murine Tcirg1oc/Tcirg1oc diseasemodel of osteopetrosiscan be rescued by gene therapy using gammaretroviral vectors to targetHSCs [14].

The aim of the present study was to rescue the phenotype of humanIMO osteoclasts by lentiviral mediated gene transfer of the TCIRG1

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cDNA. CD34+ cells from peripheral blood of five different patients withTCIRG1mutations were transduced and differentiated to mature osteo-clasts to provide the first in vitro evidence of correction of osteoclastfunction in a human genetic disease.

Materials and methods

CD34+ cell isolation, culture and expansion

Samples of peripheral blood from IMO patients or umbilical cordblood from normal deliveries were obtained after informed consentunder protocols approved by institutional ethical boards. Mononuclearcells from these cell sources were isolated on Ficoll gradient, and subse-quently CD34+ cells were separated from the mononuclear cell fractionusing MACS columns (Miltenyi Biotec, Bergisch Gladbach, Germany).Cells were cultured in SFEMStemSpanmedium(StemCell Technologies,Vancouver, BC), with the following human recombinant cytokines:M-CSF (50 ng/ml), GM-CSF (30 ng/ml), SCF (200 ng/ml), IL-6 (10 ng/ml)and Flt3L (50 ng/ml) all from R&D Systems (Minneapolis MN). CD34+

cells were plated at a density of 5 × 104 cells in 1 ml medium using24-well bacteriological plates and incubated for a week at 37 °C beforecollection and replating at a density of 1 × 105/well. Fromday 7 theme-dium was exchanged every 2–3 days by demi-depletion.

Vectors, viral production and transduction of CD34+ cells

The rescue vector used in this study, named LV-TCIRG1, contains thecDNA of human TCIRG1 inserted in a self-inactivating lentiviral vectorunder the spleen focus forming virus (SFFV) promoter, up-stream ofan internal ribosomal entry site (IRES), which is followed by the genefor enhanced green fluorescent protein (GFP). A lentiviral vector ex-pressing only GFP (LV-GFP) was used as control. Lentiviral vectorswere produced by transient transfection of the vector plasmids inhuman 293 T cells along with packaging plasmid (pCMV ΔR8.91), andenvelope plasmid (VSV-G pMDG). CD34+ cells were transduced onceon days 0–3 for 6 h in 24-well plates coated with RetroNectin (TakaraBio, Otsu, Japan) at a multiplicity of infection (MOI) of 30. After trans-duction, cells were cultured as described above and transduction effi-ciency was tested at different time points by flow cytometry analysisof cells expressing GFP. When necessary, cells received a second roundof transduction to ensure that transduction efficiency was around 40%at the end of the 2 week expansion period.

Osteoclastogenesis

After 2 weeks, the expanded cells were reseeded into 96-well plateson plastic or bovine cortical bone slices at a density of 1 × 105/wellfor cell assays, or 1.2 × 106/well on plastic in a 12-well plate forwestern blot. The cells were incubated at 37 °C and 5% CO2, in αMEMcontaining 10% serum, 100 units/ml penicillin, 100 ug/ml streptomycin,388 μg/L thymidine. They were expanded for 3 days, in the presence of50 ng/ml M-CSF. RANKL, 50 ng/ml, was added on day 3 and mediachanged every 2–3 days. After 10 days cells were fixed in 4% formalde-hyde for further analyses. Resorption was assessed by the formation ofresorption pits and the presence of CTX-I, Ca2+ and TRACP in the cellsupernatant.

Resorption biomarkers

The release of the c-terminal type I collagen fragments (CTX) frommineralized bone slices was determined using the CrossLaps for Culturekit (IDS), which was used according to the manufacturer's instructions.

The concentration of total calciumwasmeasured in culture superna-tants after resorption, by using a colorimetric assay and a Hitachi 912Automatic Analyzer (Roche Diagnostics, Basel, Switzerland) followingthe assay method validated and warranted by Roche Diagnostics [15].

Resorption pit formation

Resorption pits on the bone slices were visualized by washing themwithmilli-Qwater, followed by removal of remaining cellswith a cottonswab and then staining with Mayer's Hematoxylin for 7–8 min, follow-ed by anotherwashing inmilli-Qwater. Remaining background stainingwas removed with cotton swab if necessary. Digital micrographs wereobtained using a 20× objective and an Olympus C5050 Zoom digitalcamera mounted on an Olympus BX-60 microscope using the Cell-Asoftware (Olympus, Center Valley, PA). The resorbed area was mea-sured using NewCAST software (Visiopharm, Hørsholm, Denmark).

TRACP activity measurements

2–20 μl of conditioned media from 96-well cell cultures on eitherbone or plasticwere added to a 96well alongwith 80 μl freshly preparedreaction buffer (0.33 M acetic acid, 0.167% Triton X-100, 0.33 M NaCl,3.33 mM EDTA at pH 5.5, 1.5 mg/ml of ascorbic acid, 7.66 mg/ml ofNa2 tartrate, 3 mg/ml of 4-nitrophenylphosphate). The reaction was in-cubated at 37 C for 1 h in the dark, and then stopped by adding 100ul of0,3 M NaOH. Colorimetric changes were measured at 405 nm with650 nm as reference using a Spectramax M5 ELISA reader.

TRACP staining

After differentiation osteoclasts were fixed in 4% formaldehyde for20 min and stained for TRACP using the leukocyte acid phosphatasekit (Sigma-Aldrich, St. Louis, MO). Digital micrographs were obtainedusing a 20x objective and an Olympus C5050 Zoom digital cameramounted on an Olympus BX-60 microscope using the Cell-A software(Olympus).

Quantitative RT-PCR

RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden,Germany) and reverse-transcribed with random primers and Super-script III (Invitrogen, Burlington, Canada). Real-time PCR was performedon a LightCycler (Roche Diagnostics) using standard conditions withcDNA equivalent to 10 ng RNA and 1:20000 dilution of SybrGreen I[14]. The following primers were used: ACTIN: Fw 5′-CCATTGGCAATGAGCGGTT-3′; Rv 5′-GCGCTCAGGAGGAGCAA-3′; TCIRG1: Fw 5′-CAGCTCTTTCTGCCCACAG-3′; Rv 5′-CTGCAGGAAGGTGAAGGTCT-3′(NCBIReference Sequence: NM_006019.3). Differences in gene expressionwere calculated using the 2−ΔΔCt method.

For provirus copy number determination DNA was isolated fromtransduced CD34+ cells and from four cell lines containing one lentiviralcopy inserted. Quantitative PCRwas performed as described in previousparagraph using primers specific for HIV-1 based lentiviral vectors pro-vided in the Lenti-X Provirus Quantitation Kit (Clontech Laboratories,Mountain View, CA). Results from transduced samples were comparedto the results from the cell lines and then corrected by the transductionefficiency.

Immunoblotting

Immunoblotting was performed as previously described [16]. Cellswere harvested into RIPA lysis buffer. Protein concentrationsweremea-sured using the Bio-Rad proteinmeasurement assay (Bio-Rad, Hercules,CA). 15 μg of total protein in SDS sample buffer were loaded onto a 7.5%SDS-PAGE gel, followed by blotting onto a nitrocellulose membrane.Membranes were then blocked in TBS, 0.1% tween 20 with 5% skimmilk powder for 1 h at room temperature, followed by incubationwith primary antibody overnight at 4 °C in TBS, 0.1% tween 20, 5%skim milk powder, with the following dilutions: mouse anti-TCIRG1(Abnova, Taipei, Taiwan) 1:1000, mouse anti-Cathepsin K (Millipore,Billerica, MA) 1:1000, rabbit anti-GFP (abcam, Cambridge, UK) 1:5000,

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and p38MAPK (Cell Signaling Technology, Danvers, MA) 1:1000, whichpreviously has been shown to be a good reference [16]. The blots werethen washed 3 × 10 min and incubated with the corresponding HRP-conjugated secondary antibody for 1 h at room temperature followedby 3 × 10 min wash in TBS buffer. Blots were developed using the ECLkit (GE Healthcare, Waukesha, WI).

Cytochemistry

After fixation cells were blocked 30 min in TBS + 0.1% tween 20with 0.5% casein. They were then incubated with phalloidin (1:100,5 mg/ml stock) + TBS/Cas followed by 3 consecutive washing stepsin TBS + 0.1% tween 20. Cells transduced with LV-GFP were followedin wells during differentiation. Digital micrographs were obtainedusing a 20× objective and an Olympus C5050 Zoom digital cameramounted on an Olympus BX-60 fluorescence microscope using theCell-A software (Olympus). For antibody staining, sorted bone marrowderived CD34+ cells were differentiated into osteoclasts on glasslidesin 12-well plates. At maturation cells were fixed and then blocked inTBS + 0.1% Triton X-100 with 0.5% casein as described above. Cellswere incubated with primary antibody antibody overnight, followedby washing as described above and a 1 h incubation with secondaryantibody. Antibody binding was visualized using a DAB chromogen.Antibodies used were; primary: chicken anti-GFP 1:500 (Millipore),Secondary; goat anti-chicken antibody 1:400 (Invitrogen).

FACS Analysis of in vitro expanded cells

Cells were incubated on ice for 30 minwith the following antibodies:CD34-APC (BD Biosciences, San Jose, CA), CD14-FITC (BD Biosciences),CD11b-APC (abcam), c-Fms-PE (Santa Cruz Biotechnology, Santa Cruz,CA). Cells were then washed and suspended in PBS 2% FCS. Analysiswas performed using a FACSCanto II (BD Biosciences). The binding ofM-CSF to its receptor c-Fms can result in receptor internalization [17],therefore, prior to staining, cells were fixed and permeabilized using aCytofix/cytopermkit (BDBiosciences), such that surface and intracellularc-Fms staining could be assessed.

NSG-mice and transplantations

Breeding pairs of immunodeficient NOD-scid IL2rγ null (NSG) micewere obtained from Charles River Laboratories, Sulzfeld, Germany. Themice were maintained in the conventional animal facility at the Bio-medical Centre, University of Lund. All experiments were performedaccording to protocols approved by the local animal ethics committee.

10-week old NSG mice were sub-lethally irradiated with 300 cGyusing a 137Cs irradiator and transplanted 6 h later with 5 × 104 trans-duced CB CD34+ cells or 105 non-transduced or transduced IMOCD34+ cells by tail vein injection. Themicewere treatedwith ciproflox-acin in the drinking water for 2 weeks to avoid post-transplantationalinfections. Peripheral blood was harvested at different time points andafter termination of the mice with CO2, bone marrow cells wereharvested by crushing the femora with a mortar.

FACS analysis of cells from transplanted NSG mice

Peripheral blood and bone marrow of transplanted NSG mice wasanalyzed for human reconstitution by determining the percentage ofCD45-APC and for transduction efficiency by determining the percent-age of GFP+ cells. For lineage analysis, the cells were stained with anti-bodies directed against CD14-PE (BD Biosciences, San Jose, CA), CD33-PeCy7 (BD Biosciences), CD19-Pecy5 (BioLegend), and CD3-PE (BDBiosciences).

Statistics

Statistical analysis was performed either with unpaired two-sidedStudent's t-test or 1-way ANOVAwith a Bonferroni post-test. * indicatesp = 0.05; ** indicates p = 0.01; *** indicates p = 0.001.

Results

CD34+ cells from IMO patients generate osteoclasts with impairedresorption capacity

It has been previously described that osteoclasts can be generatedin vitro from CD34+ cells isolated from G-CSF mobilized peripheralblood [18] andwe applied the same culture protocol to CD34+ cells iso-lated from cord blood andperipheral blood of IMOpatients. Thepatientsin this study all carry mutations in the gene encoding TCIRG1 and theclinical features and mutations are described in Table 1, S1 and S2.CD34+ cells from these patients could be isolated without need for mo-bilization as they have high levels (approximately 3%) of circulatingprogenitors [19] (Table 1), whereas control cells were isolated fromcord blood because it is not possible to harvest circulating CD34+ cellsfrom healthy donors without mobilization with drugs like G-CSF orPlerixafor. The protocol includes two parts, one expansion period of2 weeks followed by a differentiation phase of 10 days. After 2 weeksof culture the cells had expanded approximately 500-fold (Fig. 1A)and CD34 expression was gradually lost while 40% became CD14+

(Fig. S1). Cells were subsequently differentiated for 10 days on boneslices with M-CSF and RANKL to mature osteoclasts. Just as osteoclastsderived from cord blood CD34+ cells, IMO CD34+ cells developed in anormal way morphologically and fused into large multinucleated cells,expressing the osteoclastmarker TRACP (Fig. 1B). Osteoclastmorpholo-gy was investigated by visualizing the presence of the actin ring, and asshown in Fig. 1, morphology of the IMO osteoclasts appeared normal(Fig. 1B). IMO TRACP activity was significantly higher compared to con-trols (Fig. 1C). IMO osteoclasts grown on bone slices failed to resorbbone as shown by lower calcium and CTX-I release (Fig. 1C). Asexpected the formation of resorption pits was also severely impaired(Fig. 1B). Expression of TCIRG1 protein was not detectable in IMO oste-oclasts, while the expression of the osteoclast specific marker cathepsinK was comparable to cord blood derived osteoclasts, suggesting thatIMOosteoclasts develop normally despite the lack of the TCIRG1 protein(Fig. 1D). Thus, although the morphology is similar to controls, osteo-clasts derived from IMO patients display impaired bone resorptionability.

Transduction and differentiation of CB CD34+ cells results in high levels ofGFP+ osteoclasts

To restore the resorptive function of IMO osteoclasts we constructeda SIN lentiviral vector expressing TCIRG1 driven by a SFFV promoterfollowed by an IRES and GFP (named LV-TCIRG1). A vector expressingonly GFP (LV-GFP) was utilized as control (Fig. S2A). These vectorswere used for transduction of CD34+ cells from IMO patients and nor-mal cord blood. The cells were transduced once at the beginning ofthe culture, between day 0 and day 3, then if necessary again at day 7,(Fig. S2B), to obtain a transduction efficiency of approximately 40%(Fig. 2A). The provirus copy number in transduced cells was determinedby qPCR and found to be in the range of 4 to 9.5 copies per GFP-positivecell. Transduced CB CD34+ cells developed normally into GFP-positivemultinucleated osteoclasts that resorbed bone and expressed TRACP(Fig. S3). A clear majority of osteoclasts were GFP-positive by fluores-cencemicroscopy at the endof the culture demonstrating a stable trans-duction. The GFP intensitywas generally lower than in themononuclearcells most likely due to fusion of GFP+ and GFP− cells (Fig. S3C), con-firmed by the observation that when cells were sorted for GFP after

Table 1Genetic analysis and clinical features of IMO patients in the study.

Case No. Origin Sex Age (years) at diagnosis Age (years) at sampling Outcome (survival-years) %CD34+ cells in PB TCRIG1mutation Status

1 Germany m 0 0.1 Alive 3.1 delTG (aa326) G N A (-1 SA) Het2 Costa Rica f 0.8 1.1 Dead (2.4) 1.6 G N A (G405R) Hom3 Costa Rica f 0.2 12.2 Dead (12.9) 2.8 G N A (G405R) Hom4 Costa Rica m 1.4 10.2 Dead (12.2) 2.1 G N A (G405R) G N T (R444L) Het5 Germany f 0.4 0.5 Alive 3.3 8590C N T (Q433X) Hom

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one week of expansion, they differentiated into osteoclasts which allexpressed GFP at a high intensity (Fig. S3).

Transduction of IMO CD34+ cells with LV-TCIRG1 results in restoredTGIRG1 protein expression in mature osteoclasts

qRT-PCR analysis was performed to evaluate the relative mRNAlevels of TCIRG1 at day 14 of expansion of transduced CD34+ cells(Fig. 2B). mRNA levels of TCIRG1 in IMO cells transduced with LV-TCIRG1 were about 10 fold higher than in untransduced cord bloodcells. TCIRG1 protein was expressed in the mature rescued osteoclastsat day 10 of osteoclast culture as determined by immunoblot (Fig. 2C)whereas there was no or very low expression in non-transduced andLV-GFP transduced IMO cells.

Fig. 1. CD34+ cells from the peripheral blood of IMO patients generate osteoclasts with impaireand differentiated into osteoclasts for 10 days in the presence ofM-CSF and RANKL. (A) Cell expculture as described inMaterials andMethods. (B) Upper panel, TRACP stain of CD34+ derivedMiddle panel, phalloidin stain of f-actin rings in IMO or cord blood CD34+ derived osteoclastsbone slices from either cord blood or IMO CD34+ derived osteoclasts grown 10 days. (C) TRAshown as mean ± SEM (D) IMO or cord blood derived CD34+ cells were differentiated into orun on lysates using anti-TCIRG1 (in-house) 1:1000, anti-Cathepsin K (Millipore) 1:1000, anti-rials and Methods. Statistical analysis was done using an unpaired two-sided Student's t-test. *

Restored resorptive function of osteoclasts from IMOpatients after lentiviralmediated TCIRG1 gene transfer

Transduced IMO and cord blood CD34+ cells were differentiatedinto osteoclasts on bone slices, and the ability of the cells to developinto osteoclasts was verified by assessing TRACP secretion whereastheir ability to resorb bone was evaluated by measuring calcium andCTX-I release into the medium. The LV-TCIRG1 transduced IMO osteo-clasts showed a lowered amount of TRACP secretion compared to bothuntransduced and LV-GFP transduced IMO cells (Fig. 3A and Table 2).A significant increase in both calcium and CTX-I levels was observed(Figs. 3B, C and Table 2) in CD34+ derived IMO osteoclasts transducedwith the rescue vector compared to non- or LV-GFP transduced IMO os-teoclasts, indicating an increase in resorptive activity and at least partialrestoration of function. We observed that the resorption of cells

d resorption capacity. CD34+ cells were expanded for 2 weeks, then seeded on bone slicesansion of normal cord blood cells compared to IMO cells assessed at 1 week and 2 weeks ofosteoclasts from either cord blood or IMO patients, cultured on bone and fixated at day 10.fixated on bone at day 10. Lower panel, hematoxylin staining of resorption pits on bovineCP activity, calcium concentration and CTX-I in the conditioned media at day 10. Data aresteoclasts on plastic for 10 days in the presence of M-CSF and RANKL. Western blot wasp38 (Cell Signaling Technology) 1:1000. All imaging was performed as described in Mate-indicates p = 0,05; ** indicates p = 0,01; and *** indicates p = 0,001.

Fig. 2. Expression of TCIRG1 in osteoclasts from IMO patients. (A) The graph shows the average transduction efficiency of all five patient samples (n = 5) and cord blood controls (n = 2)after 2 weeks of expansion. The cells were transduced as described in the experimental design in Fig. S2. Data are shown asmean ± SEM. (B) mRNAwas isolated after 14 days of expan-sion, RT-qPCR was performed and the graph shows the average relative TCIRG1 levels of patient samples (n = 3) and cord blood controls (n = 2). Data are shown as mean ± SEM.(C) Western blot analysis was performed on lysates from mature osteoclasts after 10 days of differentiation. C = untransduced control; G = LV-GFP; T = LV-TCIRG1.

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transduced with LV-TCIRG1 was 69–96% of that of osteoclasts derivedfrom normal CD34+ cord blood cells based on calcium release and 34–113% based on CTX-I levels (Fig. 3, Table 2). To further evaluate the ef-fect of lentiviral gene transfer of TCIRG1 into IMO-cells bone sliceswere stained with Mayer's hematoxylin and formation of resorptionpits visualized. After 10 days of differentiation the mature IMO osteo-clasts generated from cells transduced with LV-TCIRG1 had formed ahigh number of clearly visible pits, whereas on the bone slices withIMO cells transduced with LV-GFP they were almost absent (Fig. 4A).Quantification of the pit areas showed a rescue of approximately 59%in the LV-TCIRG1 group when compared to osteoclasts generated fromcord blood (Fig. 4B). This establishes that the introduction of non-mutated TCIRG1 into IMO osteoclasts reconstitutes their ability to acid-ify resorption lacunae and degrade bone.

Long-term engraftment of transduced CB and IMO CD34+ cells in NSGmice

To establish that the circulating CD34+ cells in IMO patients haverepopulating capacity and that these repopulating cells can be trans-duced with our vectors 10 week old NSG mice were transplantedwith transduced or non-transduced CB or IMO CD34+ cells (Table 3).7–32 weeks post transplantation the mice were sacrificed and bonemarrow analyzed for human reconstitution and level of GFP-marking(Table 3). In all transplanted mice human CD45+ cells were observed,showing that PB IMO CD34+ cells have the capacity to engraft in NSG-mice. In addition, GFP-marked cells were found in all mice transplantedwith transduced cells either from CB donors or IMO patients,establishing gene transfer to NSG-repopulating cells with our vectorswithout any signs of toxic effect of the therapeutic vector (Table 3). Spe-cifically, all four mice transplanted with LV-TCIRG1-transduced IMOCD34+ cells exhibited GFP marking despite low transduction efficiencyin vitro (10%) in this experiment. No differences in lineage distribution

of bone marrow cells harvested from mice transplanted with humanCD34+ cells transduced with LV-GFP or LV-TCIRG1 was observed, indi-cating that LV-TCIRG1 does not skew differentiation potential of trans-duced CD34+ cells (data not shown).

Discussion

The aim of the present study was to rescue the phenotype of humanIMOosteoclasts by lentiviralmediated gene transfer of the TCIRG1 cDNAto CD34+ cells. CD34+ cells from normal mobilized peripheral bloodhave been shown capable of high levels of proliferation and differentia-tion into immature macrophages with expression of pre-osteoclastmarkers in the presence of specific cytokines [18]. Furthermore it hasbeen shown that cord blood CD34+ cells subjected to lentiviral trans-duction retained high levels of marker gene expression after osteoclastdifferentiation [20]. By applying the expansion protocol mentionedabove to CD34+ cells fromnormal cord blood and fromunmobilized pe-ripheral blood of IMO patients we were able to obtain high amounts ofcells that after further differentiation with M-CSF and RANKL exhibitedboth molecular and phenotypic osteoclast markers as also observed byRamnaraine et al. [20]. Osteoclasts derived from normal cord bloodwere capable of resorbing bone effectively in vitro while osteoclastsfrom IMO patients exhibited strongly impaired resorption as expected[9,21].

For the gene transfer experiments we chose a SIN lentiviral vectorfor efficient and stable gene expression, and also because these vectorsfor the future prospect of clinical gene therapy have shown to be lessprone to insertional mutagenesis compared to gammaretroviral vectors[22,23]. Due to the limited patient material available we chose a vectorwith the SFFV-promoter to ensure a sufficiently high level of transgeneexpression.

Fig. 3. Lentiviral transduction of TCIRG1 into IMO CD34+ cells results in restoration of resorptive function in differentiated osteoclasts. CD34+ cells from patient 4 were transduced andcultured as described in Materials and methods. Media was collected every 2–3 days during differentiation and tested for TRACP, Ca2+ and CTX-I. Cord blood from healthy donors wasused as positive controls and IMOpatient cells, either untransduced (IMOcontrol) or transducedwith LV-GFP vector,were usedas negative controls. (A)Osteoclast formationwas assessedon day 10 bymeasuring TRACP activity in themedia. (B) Breakdown of inorganic bonematrixwas evaluatedmeasuring calcium release into themedia between day 7 and 10. (C) Organicbone resorption was assessed on day 10 by measuring CTX-I concentration in the supernatant. (D) The CTX-I/TRACP ratio represents release of CTX-I per osteoclast in culture. Statisticalanalysis was done using 1-way ANOVA with a Bonferroni post-test. * indicates p = 0,05; ** indicates p = 0,01; and *** indicates p = 0,001.

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When normal CD34+ cord blood cells were transducedwith the GFPvector and then differentiated into functional osteoclasts the number ofGFP positive cells increased dramatically due to the fusing nature of os-teoclast maturation, and constituted a clear majority of multinucleatedosteoclasts. However, the intensity of GFP expression in the individualosteoclasts was lower, likely due to the fusion of GFP+ and GFP−mono-cytic cells. When sorting GFP positive cells, they differentiated into

Table 2Functionality of osteoclasts derived from transduced IMO patient CD34+ cells. All valuesare based on analysis from resorption assays at day 10 of osteoclast differentiation.TRACP, an osteoclast marker, is shown as percentage of CB GFP. Calcium and CTX-I, boneresorption markers, are shown as percentage of CB GFP. IMO GFP is deemed backgroundlevels and therefore subtracted from the shown calcium and CTX-I percentages. Pitscore, a histological assessment of bone resorption, is shown aspercentage of pit formationgenerated by osteoclasts derived from CB CD34+ cells transduced with LV-GFP. Meanvalues are based on percentages of all 5 patients. Total mean values are calculated fromthe individual mean of each patient.

Patient TRACP % of CBGFP ± SEM

Calcium % of CBGFP ± SEM

CTX-I % of CBGFP ± SEM

Pit Score % of CBGFP ± SEM

LV-GFP LV-TCRIG1 LV-TCRIG1 LV-TCRIG1 LV-TCRIG1

1 159 ± 16 79 ± 7 93 ± 15 34 ± 4 14 ± 42 137 ± 19 159 ± 23 92 ± 15 51 ± 5 28 ± 113 200 ± 15 190 ± 12 84 ± 7 55 ± 3 91 ± 124 204 ± 4 138 ± 8 96 ± 5 102 ± 10 80 ± 135 197 ± 14 116 ± 8 69 ± 10 113 ± 8 63 ± 21Mean 179 ± 13 136 ± 19 86 ± 5 71 ± 15 59 ± 9

osteoclasts which all expressed GFP at a high intensity, confirming thishypothesis. The fusion of transduced and non-transduced cells couldprove to be beneficial when transducing patient cells with rescue vec-tors, as it may not be necessary for all the monocytic cells that fuse tobe transduced to obtain a functional osteoclast [24].

CD34+ cells from peripheral blood of five patients with differentTCIRG1 mutations were transduced and qRT-PCR analysis showedoverexpression of TCIRG1 at the mRNA level. After differentiationto mature osteoclasts western blot analysis showed the presence ofthe a3 subunit after rescue whereas it was absent or very low in non-transduced or LV-GFP-transduced IMO cells.

The main objective of the current work was to assess the functionalrestoration of osteoclasts differentiated from the transduced cells. Theability of osteoclasts to resorb bone was evaluated by measuring calci-um release and release of the resorption marker CTX-I into the media.Resorption was on average 86% that of osteoclasts derived from cordblood cells based on calcium release and 71% based on CTX-I. As furtherconfirmation of the rescued phenotype, mature IMO osteoclasts trans-duced with LV-TCIRG1 showed a high amount of clearly visible resorp-tion pits when differentiated on bone slices whereas in IMO controls,transduced with LV-GFP, pits were in most cases completely absent inline with previous studies [9]. This clearly establishes that the introduc-tion of non-mutated TCIRG1 into IMO osteoclasts restores their ability toacidify the resorption lacunae and resorb bone in vitro. Furthermore, inthe IMO cells the TRACP levels are increased, a phenomenonwhich pre-viously has been shown to be related to increased survival of the osteo-clasts due to their lowered resorption activity [21,25,26]. Importantly,

Fig. 4. Lentiviral transduction of TCIRG1 into IMO CD34+ cells results in formation of resorption pits in bovine bone slices by differentiated osteoclasts. (A) At day 10 of osteoclast differ-entiation boneslices were stained for resorption pits as described in materials and methods. Formation of resorption pits was visualized in a microscope with a 4× objective and are rep-resentative of 3 different bone slices per condition. (B) Pit formation was quantified by assessing the area resorbed on bone slices using NewCAST software from VisioPharm. Results areshown as themean area resorbed by osteoclasts derived fromCD34+ cells from patient 1–5 expressed as percentage of area resorbed by osteoclasts generated from CB CD34+ cells trans-duced with LV-GFP.

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Table 3Engraftment of transduced CB and IMO CD34+ cells in immunodeficient NSG-mice.10-week old NSGmice were irradiated with 300 cGy and transplanted with 5 × 104 transduced CB CD34+ cells or 105 non-transduced or transduced IMO CD34+ cells by tail vein injec-tion. At the indicated time points mice were sacrificed and bonemarrow analyzed for percentage of human CD45+ cells and level of GFP marking. Mean and range shown. N.D. is not de-termined. N.A. is not applicable.

Cell transplanted and vector used % in vitro transduction efficiency No. of mice Time of analysis (weeks post tx) %CD45+ cells in BM %GFP+ cells in CD45+ population

CB CD34+ LV-GFP 46 2 7–32 51 (33–70) 33.5 (16–51)CB CD34+ LV-TCIRG1 N.D. 5 13–29 28.8 (1.5–75.3) 15.8 (2.7–26)IMO CD34+ Untransduced N.A. 4 12 36.4 (22.5–59) N.A.IMO CD34+ LV-TCIRG1 10 4 12 47.2 (43.4–50) 6.7 (2.3–18.4)

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this process is not specific to lack of TCIRG1 as it is also observed in c-src,cathepsin K and ClC-7 deficient osteoclasts [4,27–30]. Finally, the intro-duction of the LV-TCIRG1 corrects the increased numbers of osteoclasts,which correlates well with the correction of resorptive activity and thusthe restoration of the normal auto-regulatory loop controlling osteo-clast activity and life span [25].

The in vivo experiments using the NSG mouse model show twothings. First, IMO CD34+ cells fromPB can engraft in this xenotransplantmodel and that is in line with the observation that they can be used asback-up in the clinical transplantation setting if graft failure occurs[19]. Second, the progeny of CB and IMO CD34+ cells transducedwith the TCIRG1 expressing vector were observed in NSG mice up to32 weeks, indicating gene transfer to NSG-engrafting cells withoutany indication of significant vector mediated toxicity. The limited avail-ability of patient cells precluded more detailed analysis in this modeland in vivo correction of osteoclasts was not possible to show as thesecells do not develop in this xenotransplant model due to the speciesspecificity of M-CSF [31].

For future development and application of a clinical gene therapyprotocol for treatment of IMO a crucial question is what level of correc-tion of osteoclast function is needed in vivo to reverse the disease phe-notype. In the present study lentiviral gene transfer of TCIRG1 resultedin an average transduction efficiency of 45% andbone resorptionwas re-stored to 69–96% and 34–113% of that of osteoclasts derived from nor-mal CB CD34+ cells, as determined by calcium and CTX-I releaserespectively. In the severe mouse model of IMO, the Tcirg1oc/Tcirg1oc

mouse, we previously showed that transplantation of gene correctedcells could completely reverse the disease, even though the in vitrobone resorption capacity of these cells was only 10% of wild type cells[14]. Furthermore, we were also able to show that transplantation ofwild type cells in a non-myeloablative setting, resulting in an engraft-ment level of only 4–5%, was sufficient to correct the disease [32]. Ifwe consider the Tcirg1oc/Tcirg1oc mouse a relevant model for IMO inhumans, these data, taken together, make it likely that the level of cor-rection obtained in the present study would be sufficient to ameliorateIMO symptoms also in humans. However, we realize that for gene ther-apy of IMO to be considered in a clinical setting we must substitute theSFFV-promoter in our vector with mammalian and/or tissue specificpromoters and evaluate the level of rescue that can be obtained.

In summary, in this report we provide the first evidence of lentiviral-mediated correction of a genetic disease involving the osteoclast lineage,supporting further development of hematopoietic stemcell targeted genetherapy of not only IMO but also of other diseases affecting osteoclasts.

Author disclosure statement

No competing financial interests exist.

Acknowledgements

WethankChristopher BaumandAxel Schambach (HannoverMedicalSchool, Germany) for providing us with the lentiviral vector backbone.IM received funding by Foundation BLANCEFLOR Boncompagni-Ludovisi, née Bildt. CST received funding fromNordforsk. CF is supported

by a PhD fellowship from European Calcified Tissue Society. KH issupported by the Danish Research Foundation (Den DanskeForskningsfond). JR is supported by grants from The Swedish ChildhoodCancer Foundation, a Clinical Research Award from Lund University Hos-pital, Magnus Bergvalĺs Foundation, the Georg Danielsson Foundationand The Foundations of Lund University Hospital. AS is partially support-ed by grants of the EU (ERARE initiative, project OSTEOPETR). AV is sup-ported by the RF2009-1499542 project. The Lund Stem Cell Center issupported by a Center of Excellence grant in life sciences from the Swed-ish Foundation for Strategic Research. The funders had no role in studydesign, data collection and analysis, decision to publish, or preparationof the manuscript.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bone.2013.07.026.

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