WO2019232426A1 - Methods and compositions for the treatment of osteopetrosis - Google Patents

Abstract

Disclosed herein are methods and compositions for treating, preventing, or ameliorating osteopetrosis. In some embodiments, the present technology relates to administering a composition comprising a therapeutically effective amount of engineered monocytic cells or wild-type monocytic cells from a healthy donor to a subject suffering from or at risk for osteopetrosis.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF

OSTEOPETROSIS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U.S. Provisional Patent Application No. 62/679,553, filed on June 1, 2018, the contents of which are hereby incorporated by reference in their entirety.

STATEMENT OF FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under CA008748, AI130345 and HL 138090 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

[0003] The present technology generally relates to methods and compositions for treating, preventing, or ameliorating osteopetrosis. More specifically, the present technology relates to administering a composition comprising a therapeutically effective amount of engineered monocytic cells or wild-type monocytic cells from a healthy donor to a subject suffering from or at risk for osteopetrosis.

BACKGROUND

[0004] The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art.

[0005] Osteoclasts are multinucleated giant cells that resorb bone, ensuring development and continuous remodeling of the skeleton and the bone marrow hematopoietic niche.

Defective osteoclast activity leads to osteopetrosis and bone marrow failure, while excess activity can contribute to bone loss and osteoporosis. Osteopetrosis can be partially treated by bone marrow transplantation in human and mice, in accordance with osteoclasts hematopoietic origin, and studies suggesting that they develop by fusion of hematopoietic stem cell (HSC)-derived monocytic precursors in the presence of CSF1 and RANK-Ligand. However, the developmental origin and lifespan of osteoclasts, and the mechanisms that ensure maintenance of osteoclast function throughout life in vivo remain largely unexplored. Moreover, there is a need to develop alternative therapeutic approaches to the treatment of osteopetrosis as the current treatment by bone marrow transplantation is plagued by an approximate 48% overall survival at 6 years.

SUMMARY

[0006] In one aspect, the present disclosure provides a method for treating or preventing osteopetrosis in a subject in need thereof, the method comprising administering a

composition comprising a therapeutically effective amount of monocytic cells from a healthy donor to the subject. In some embodiments, the subject is characterized by decreased expression of one or more of CA2 , CLCN7 , CTSK , CSF1R , IKBKG, ITGB3 , OSTM1 , PLEKHM1 , TCIRG1 , TNFRSF11A, and TNFSF11, as compared to the monocytic cells of the donor. In some embodiments, the osteopetrosis comprises one or more of stunted growth, skeletal deformity, increased likelihood of bone fracture, anemia, recurrent infections, hepatosplenomegaly, facial paralysis, abnormal cortical bone morphology, abnormal form of vertebral bodies, abnormal temperature regulation, abnormality of the ribs, abnormality of vertebral epiphysis morphology, bone pain, cranial nerve paralysis, craniosynostosis, hearing impairment, and hypocalcemia. In some embodiments, the composition is formulated for intravenous administration by injection, infusion, or transfusion. In some embodiments, the subject is a mammal. In some embodiments, the mammalian subject is a human. In some embodiments, the subject is characterized by a cathepsin K deficiency.

[0007] In one aspect, the present disclosure provides a method for treating or preventing osteopetrosis in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of monocytic cells engineered to express one or more genes selected from CA2, CLCN7 , CTSK , CSF1R, IKBKG, ITGB3, OSTMl, PLEKHM1 , TCIRG1 , TNFRSF11A , and TNFSF11. In some embodiments, the osteopetrosis comprises one or more of stunted growth, skeletal deformity, increased likelihood of bone fracture, anemia, recurrent infections, hepatosplenomegaly, facial paralysis, abnormal cortical bone morphology, abnormal form of vertebral bodies, abnormal temperature regulation, abnormality of the ribs, abnormality of vertebral epiphysis morphology, bone pain, cranial nerve paralysis, craniosynostosis, hearing impairment, and hypocalcemia. In some embodiments, the composition is formulated for intravenous administration by injection, infusion, or transfusion. In some embodiments, the subject is a mammal. In some embodiments, the mammalian subject is a human. In some embodiments, the subject is characterized by a cathepsin K deficiency. In some embodiments, the monocytic cells are obtained from the subject.

[0008] In one aspect, the present disclosure provides a donor monocytic cell line engineered to express one or more genes selected from CA2 , CLCN7 , CTSK , CSF1R , IKBKG, ITGB3, OSTM1, PLEKHM1 , TCIRG1 , TNFRSF11A , and TNFSF11 , wherein the one or more genes is operably linked to a heterologous nucleic acid to form a chimeric nucleic acid construct. In some embodiments, the heterologous nucleic acid encodes a selectable marker. In some embodiments, the selectable marker is a bioluminescent protein, a fluorescent protein, a chemiluminescent protein, a xanthine-guanine phosphoribosyl transferase gene (gpt), or any combination thereof. In some embodiments, the heterologous nucleic acid encodes one or more control sequences suitable for directing expression of the one or more genes in a monocytic cell. In some embodiments, the one or more control sequences comprises a promoter. In some embodiments, the donor cells comprise a vector encoding one or more genes selected from CA2, CLCN7, CTSK, CSF1R, IKBKG, ITGB3, OSTM1 ,

PLEKHM1, TCIRG1, TNFRSF11A, and TNFSF11. In some embodiments, the vector is a mammalian expression vector, a lentiviral vector, or transposon vector.

[0009] The technology described and claimed herein has many attributes and embodiments including, but not limited to, those set forth or described or referenced in this brief summary. It is not intended to be all-inclusive and the technology described and claimed herein is not limited to or by the features or embodiments identified in this brief summary, which is included for purposes of illustration only and not restriction. Additional embodiments may be disclosed in the detailed description below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figures 1A-1I. HSC-derived precursors are dispensable for osteoclasts and bone development. Figure 1A: Top: Representative photographs of teeth of control littermates and Csflr^Cre; CsflrKA 3-4 week old mice (right) and controls and Csflr^Cre ;Tnfrsfl lcfiF mice (left). Bottom left: Representative photographs of leg bones from controls and CsflC ⁿ‘; Tnfrsflla¹¹¹¹ mice, white arrowhead points to lack of blood cells. Right: bar graphs represent osteoclast numbers in E18.5, P7, and 4-week old CsflC ^rc; Tnfrsfl k ¹¹ mice and littermates (n=3/3), and representative hematoxylin/TRAP staining of femur sections; arrow indicates an osteoclast. Figure IB: Representative photographs of teeth of 3- 4 week old Flt3^Cre ;Tnfrsfl IcfJ¹ , Flt3^Cre Csflr^A , Vav^Cre ;Csflr^ and littermates. Figure 1C: Bone marrow CD45+ cells numbers in l'h3 ^,v; Tnfrsfl I a¹¹¹¹ and littermates at 4 (n=4/3) and 22 week old (n=4/5) and Vav^Cn';Csflr¹¹¹¹ and littermates at 4 (n=8/8) and 62 week old (n=5/8) of the indicated age. Figure ID: Osteoclast counts in femurs from Tlt3^{( r} ; Tnfrsfl I a^{,I ,} Flt3^Cre ;Cflr^ ,

Vav^{( r,} ;CsfIr¹¹¹¹ mice and littermates of the indicated ages. Figure IE: Representative hematoxylin/TRAP staining of femur sections from 22 week old Flt3^Cre ;Tnfrsfl lcfP , 62 week old Vav°^D/Cre ;CsflrFfl mice and littermates. White arrowhead points to trabecular bone.

Figure IF: Quantitative analysis of bone volume/total volume ratio of humerus or femurs from TH3^{( r} ; Tufrsfl I a¹¹¹¹ (n=4), Flt3^Cre iCsflr^fl (n=5) and controls littermates (n=8), and Vav^c, ;CsfIr¹¹¹¹ (n=5) mice and controls littermates (n=5) as determined by MicroCT and Statistical significance determined by ANOVA. Figure 1G: Expression and MFI of YFP in in TRAP⁺ multinucleated cells from Csflr^Cre ;Rosa26^LSL~YFP mice at embryonic day E16.5,

E18.5, P7, and 6 months, (n=3 per time point). For MFI and percentages, at least 100 osteoclasts were quantified per time point, genotype, displayed as a box plot, min, Ql, median, Q3 and max. Figure 1H as in Figure 1G for Flt3^Cre ;Rosa26^LSL~YFP mice at El 6.5,

El 8.5, P7, 4 weeks (4w), 3 months, and 6 months. Data are mean ± SD, circles represent individual independent biological replicates, n indicates number of mice per group. Statistical significance was analysed with GraphPad Prism using unpaired two-tailed t-tests unless otherwise indicated. Figure II: Representative confocal microscopy image of frozen sections from mice in (Figures 1G, 1H). Statistical significance was analysed with GraphPad Prism using unpaired t-tests and two-way ANOVA with Tukey’s multiple comparisons test as indicated in the figure legends. Significance was considered at p value (p) *p < 0.05; **p <

0.01; ***p < 0.001; ****p < 0.0001.

[0011] Figures 2A-2J. EMP derived osteoclasts are required for bone development.

Figure 2A: MGC number in femur anlage’s ossification centers from El 5.5 / E l 6.5 Mylr (n=6) and littermate controls (n=3). Figure 2B: Representative confocal microscopy pictures of frozen sections from Figure 1 A stained with fluorescent TRAP and TO-PRO-3. Figure 2C: Percent of TRAP+ cells expressing YFP in femur anlage from E15.5

Csflr^MerCreMer ;Rosa26^LSL~YFP mice (n=8) and Cre negative controls (n=5), pulsed at E8.5 with 4-OHT. Figure 2D: Representative confocal microscopy picture from Figure 2C. Figure 2E: Representative photographs of teeth of control littermates (Cre ), Tnfrsfl I a^{Koh ~( n}‘;( "sflr¹¹¹¹ and Tnfrsfl la^Wask~Cre CsflrFfl mice (Cre⁺). Figure 2F: Leg bones from Tnfrsfl 1 a^{1 rc};( 'sflr¹¹¹¹ mice (Cre⁺, n=6) and control littermates (Cre , n=6) at P7and 4 weeks. Arrowhead points to the color of bone. Figure 2G: Representative MicroCT scans of long bones from mice in Figure 2E (n=6 per genotype). Figure 2H: Skull length from 3 weeks old Csflr ⁷ (n=6) controls littermates/h= 12) and Tnfrsf lla^Wask~Cre CsflrdP ice (n=4) as determined by

MicroCT. Figure 21: Osteoclasts counts in bone sections from E18.5, P7, and 3-4 week old Infrsfl la^{( re} ;CsfI r^{l! 11} mice and littermate controls. Figure 2J: Numbers of bone marrow CD45+ cells determined by flow cytometry in 4 weeks old littermates (Cre , n=l3,27),

Tnfrsfl la^Koha~Cre Csflrfl^ (n=9) and Tnfrsfl la^Wask~Cre ;Csflr-f^l/fl (n=23) mice (Cre⁺). Data are mean ± SD, circles on graphs represent individual mice, n indicates the number of mice per group. Statistical significance was analysed with GraphPad Prism using unpaired two tailed t- tests. Significance was considered at p value (p) *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0012] Figures 3A-3G. In vivo dynamics of osteoclasts. Figure 3A: Parabiosis of

Csflr^Cre ;Rosa26^{LSL YFP} mouse surgically paired with a Csflr^Cre ;Rosa26^{LSL tdTomato} partner for 4 to 8 weeks. Representative (n=3) confocal microscopy image of frozen sections from the femur of a Csflr^Cre ;Rosa26^LSL~YFP partner stained with antibodies for tdTomato (red) and YFP (green), ELF97 (blue) and TOPRO-3 (grey). Figure 3B: Pie graphs represent the percentage of tdTomato positive (red), YFP positive (green) and double tdTomato/YFP positive cells (yellow) among BM mononuclear cells (MNC), Megakaryocytes (Mega) and Multinuclear Giant Cells (MGC), from parabionts paired for 1, 2, 3, 4, and 8 weeks(n=8). Figure 3C: As in Figure 3B for parabionts separated after 4 weeks and analysed 14 weeks and 24 weeks after separation (n=3). Figure 3D: Scatter plots represent the Mean Fluorescent Intensity (MFI) of individual TRAP+ MGC for YFP (Y axis) and tdTomato (X axis), and histograms represent the overlayed distribution of the MFI values for YFP and tdTomato in TRAP+ MGC at the indicated time-points. Figure 3E: Bar graph display number of nuclei per TRAP+ MGC in femurs from wild type mice at 1, 3, and 6 month of age (n=3 mice per time point). Figure 3F: Representative confocal microscopy image of an EdU-labeled nucleus in a TRAP+ osteoclast, and histogram displaying the percentages of TRAP+ osteoclast with EdU-labeled nuclei and the number of labeled nuclei per cell 72 hrs after intravenous pulse-labeling with EdU (n=5 mice). Figure 3G: A model for development and maintenance of osteoclast syncytia. Data are mean ± SD, circles on graphs represent individual mice, n indicates the number of mice per group. Statistics: two-way ANOVA with Tukey’s multiple comparisons test *p<0.05 **p<0.005. [0013] Figures 4A-G. Rescue of Osteopetrosis. Figure 4A: Bone volume over total volume (BV/TV%) of femurs from 10 week-old Cathepsin K ^/_ mice (n=3) after 6 weeks parabiosis with Cathepsin K^+/ heterozygote mice and from positive (n=4) and negative (n=4) controls parabionts, analysed by Von Kossa staining. Figure 4B: Monocyte transfer:

histograms represent percentages of tdTomato+ cells among bone TRAP+ MGC from

Csflr^Cre ;Rosa26^{LSL YFP} recipients analysed by confocal microscopy {left) 11 days (n=2) and 60 days (n=5) after i.v. transfer at age 6 week-old of 3xl0⁶ bone marrow Ly6C⁺ cells from Csflr^Cre ;Rosa26^{LSL tdTomato} donors, and percentages of tdTomato+ cells among bone marrow precursors and blood leukocytes analyzed by flow cytometry after 60 days (n=5, right).

Figure 4C: Representative high power confocal microscopy image of the femur of a recipient mouse 60 days after i.v. transfer (from Figure 4B), stained with antibodies for tdTomato and YFP, and ELF97 and TOPRO-3. Figure 4D: Representative photographs of teeth, and CT scan of leg bones from ( sflr^cr ; Csflr^fl mice (n=3) transferred with monocytic cells from Csflr^Cre; Rosa26^{LSL YFP} donors at post-natal day 5, 8, and 11, and from wt and non-transferred Csflr ^cre; Csflr fl controls. Arrows indicated the presences of teeth eruption (upper panel) and bone marrow cavity (lower panel). Figure 4E: Representative confocal microscopy image of femur from mice #3 in Figure 4D, stained with antibodies for YFP, ELF97 and TOPRO-3. Figure 4F: Histograms represent {Left) the number of TRAP+ osteoclasts number in bone sections from mice in Figure 4D and non-transferred controls and {Right) the percentage of TRAP+ cells that are YFP+ in transferred mice. Symbols represents individual mice, values are average for 3 sections per mice. At least n = 100 osteoclasts were quantified per mice. Figure 4G: Histograms represent percentages of YFP+ cells among bone marrow precursors and blood leukocytes in the recipient mice (in Figure 4D) at the time of analysis. Data are mean ± SD, circles on graphs represent individual mice, n indicates the number of mice per group. Statistical significance was analyzed with GraphPad Prism using ANOVA with Tukey’s multiple comparisons test ***p < 0.001; ****p < 0.0001.

[0014] Figures 5A-G. Csflr^{( rc}; Trfrsfl la¹¹¹¹, Csflr^A ;Csflr^Cre , and Tnfrsfl la^Cre ; Csflr^A mice are osteopetrotic. Figures 5A-B: Representative Computed Tomography scans

(nanospect CT) of 4 week old Csflr^Cre ;Tnfrsfl laf¹^ mice arrows point skull deformation, and absence of bone marrow cavity in mutant mice. Figure 5C: H&TRAP staining of bone sections of 4 week old Csflr^Cre ;Tnfrsfl lcfl^ mice showing closure of the bone marrow.

Histology of paraffin sections (5pm thickness) corroborates the phenotype from mice in Figure 5B. Figure 5D: Representative photograph of inguinal lymph nodes in Csflr^Cre ;Trfrsfl lcpF mice. Figure 5E: Representative photograph of Csflr^Cre ;Trfrsfl lcfP and littermates controls. Figure 5F: Representative computed tomography scan

reconstructions (nanospect CT) of 4 weeks-old Tnfrsfl la^(Koba)Cre; CsflrTJ¹ mice. Arrows point to skull deformation, but the presence of bone marrow cavity in mutant mice, as in Figures 5A, B. Figure 5G: Representative Computed Tomography scans of Csflr^Cre; Csflr^ mice. Arrows shows skull deformation, and absence of bone marrow cavity in mutant mice as in A,B,. CT scans and photographs are representative of >10 litters.

[0015] Figures 6A-N. Bone histology and Flow cytometry analysis of bone marrow phenotypic KSL, LT-HSCs, ST-HSCs, andMPPs in mice of indicated genotypes. Figure 6A: Young Flt3^Clc;Csf 1 r^il/n and Flt3 ^Cre; Tnfrsfl la^fl/flmice have normal long bone: H&TRAP staining of bone sections from 4 week old Flt3^Cre ;Tnfrsfl lcflJ¹ mice showing normal bone structure and bone marrow cavity. Figure 6B: LSK cell numbers in bone marrow

Flt3^Cre ;Tnfrsfl lcflF (n=3), Flt3^Cre ; Tnfrsfl lcf^/+ mice (n=2), and littermate controls (n=4) in 3- 4 week old mice and Flt3^Cre ;Tnfrsfl la¹¹¹¹ (n=5), Flt3^Cre ;Tnfrsfl kP mice (n=4), and littermate controls (n=4) in 22 week old mice. Figure 6C: H&TRAP staining of bone sections from 4 week old IΊΐ3^{( n}';CsflP^{l l!}m\ce showing normal bone structure and bone marrow cavity. Figures 6D, E: Phenotypic long-term hematopoietic stem cells (LT-HSCs) are reduced in aged but not old

(Figures 6F, G) LT-HSC are reduced in in young Csfl r ^/_ and to a lesser extent in young Tnfrsf l 1 y^{lV sklC :}Csfl ¹ Numbers per 2 femur are shown. Figure 6H: Flow cytometry analysis of F4/80+ cells in brain (microglia) and epidermis (Langerhans cells) in E18.5 Tnfrsfl la^Cre; Csfl rTJ¹ embryos and littermate controls (n=3 per group). Figure 61: H&TRAP staining of bone sections from P7

Tnfrsfl la^Cre ;CsflrFT and littermate control showing absence of the bone marrow cavity. Figure 6J: Flow cytometry plot of Fetal liver at E15.5 (B/C representative results of 3 experiments). Figure 6K: LSK numbers in bone marrow of 3-4 week old

and littermate controls (n=20) and Tnfrsfl la^Cre(koba) ;CsflrFT (n=7), Tnfrsfl la^Cre(koba) ;CsflrT^+l {n=6) and littermate controls (n=8). Figure 6L: For comparison, LSK numbers in bone marrow of 3-4 week old Csflr ^/_ (n=22)and littermate controls (n=2l). Figure 6M: Representative micrographs of femur sections from 4 week old Tnfrsfl la^Cre ;CsflrFT and littermate control stained with hematoxylin/TRAP. Figure 6N: Blood leukocytes numbers in 4 week old Tnfrsfl la^Cre ;CsflrFT mice (n=5),

Tnfrsfl la^Cre ;CsflrF⁺ mice (n=6) and littermate controls (n=l2). Circles represent individual mice, results from 3 independent experiments. Data are mean ± SD, circles on graphs represent individual mice, n indicates the number of mice per group. Statistical significance was analysed with GraphPad Prism using unpaired two tailed t-tests. Significance was considered at p value (p) *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001. LT-HSC: Lin- Kit+ Sca-l+; Kit+ Scal+ CD34- Flt3-. Phenotypic short-term hematopoietic stem cells (ST-HSC): Lin- Kit+ Sca-l+ CD34+ Flt3-. Multipotent progenitors (MPP): Lin- Kit+ Sca-l+ CD34+ Flt3+. Lin: CD3 CD19 NK1.1 Terl l9 CDl lb Grl B220.

[0016] Figures 7A-E. Bone histomorphometry in old Flt3^Cre ;Tnfrsfl lc ^, FltS^ CsflrFfl, and Vav^Cre CsflrFflmice and control littermates. Figure 7A: Representative Micro CT pictures , genotype is indicated. Figure 7B: Bone length, connectivity density (Conn density), Trabecular Number (Tb.N.), and Trabecular Spacing (Tb.Sp.) analyzed by microCT in aged Flt3^Cre ;Tnfrsfl IcflF (n=4), l· Ί 13 ^{( re} ; ( is// r^¹ ( n =4 ) and control littermates (n=7). Figure 7C: Bone histomorphometry as in Figure 7B, in Vav^Cre; Csflr^fl mice and control littermates (n=5). Figures 7D, E: Dynamic bone histomorphometry in aged Flt3^Cre ;Tnfrsf l 1 a^il/nand Flt3^Cre ;Csf 1 r^il/n mice using in vivo calcein labeling. Representative micrographs of calcein labeling (green) of femura of mice from the indicated genotypes and ages (Figure 7D). Scale bars represent 200pm (top) and 50pm (bottom). Quantification of calcein labeling by fluorescence microscopy of mineralized surface/bone surface (MS/BS), mineral apposition rate (MAR), and bone formation rate/bone surface (BFR/BS) in aged Flt3^Cre ;Tnfrsfl lcf^ (n=5), 1Ίΐ3^{( re};( sfl r^,l/),(n=3 ) and control littermates (h=10) Data are mean ± SD, circles on graphs represent individual mice, n indicates the number of mice per group. Statistical significance was analysed with GraphPad Prism using unpaired two tailed t-tests.

Significance was considered at p value (p) *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0 0001

[0017] Figures 8A-E. Colonization of the bone marrow by Csflr+ and Flt3+

hematopoietic cells. Figure 8A: Representative confocal microscopy images of frozen sections from Flt3^Cre ;Rosa26^LSL~YFP and Csflr^Cre ;Rosa26^LSL~YFP mice analyzed at El6.5 (n=3). Figure 8B: YFP labeling efficiency in Flt3^Cre ;Rosa26^LSL~YFP mice analyzed by flow cytometry in the indicated cell populations, and by confocal microscopy on frozen bone sections at the indicated time (Right). Insets show YFP expression on individual osteoclasts. YFP: anti-GFP antibody, TRAP: ELF97 fluorescent substrate, TO-PRO-3: nuclear stain. Figure 8C: YFP labeling efficiency in Csflr^Cre ;Rosa26^LSL~YFP mice analyzed as in Figure 8A. Data from Figures 8A, B are representative of at least 3 experiments by time point and genotypes. Circles represent individual mice. Genetic lineage tracing of osteoclasts in ossification centers using Csflr^MenCreMer ; Rosa26^{LSL YFP}. Representative high power confocal microscopy images from embryonic femurs showing MGCs in primary ossification centers from Csflr^MenCreMer ;Rosa26^{LSL YFP} . Figure 8D: El 8.5 embryos pulsed with 4- hydroxytamoxifen at E8.5 showing YFP expression in MGCs after Cre recombination and quantitated as MFI (right bar graph) from Cre+ (n=8) and Cre-(n=4), and Figure 8E unpulsed controls showing the lack of YFP in Cre+ (n=4) and Cre- (n=4). Sections were labeled with antibodies against YFP, TRAP (ELF97 substrate) and TO-PRO-3 as a nuclear stain.

[0018] Figures 9A-D. Tnfrs 1 la^(wask)Cre knock-in mice allow deletion of target genes in fetal macrophages, but not definitive HSC and their progeny in blood and tissues, while Fav^cre mice allow deletion of target genes in definitive HSC, but not fetal macrophages . Figure 9A: Bar graphs indicate percentages of eYFP expression by flow cytometry in cells from

Tnfrsfl la^Cre ;Rosa26^LSL~YFP in the indicated cell types, organs, and time points. Data represent 3 independent experiments n numbers indicated on x axis. Figure 9B: Lineage tracing in the fetal liver of Vav^cre+;tdRFP^wt/kl mice n numbers indicated on x axis. Figure 9C:

Representative molecular analysis of Csflr deletion in purified bone marrow HSPC from 62 weeks old Vav^cr' ;CsfI r^llJl m\ce and controls (n=5). Figure 9D: Representative photograph of teeth from 3 week old R.26-CreER^T2+ Csflr^ pulsed with Tamoxifen at E10.5 (n=3 mice from 3 independent litters). FL: Fetal Liver, HSPC: hematopoietic stem and progenitor cell, LT-HSC: Long-term Hematopoietic Stem Cells, MPP: Multipotent progenitors, PMN:

polymorphonuclear cells, mono: monocytes, T: T cells, B: B cells, PEC: peritoneal exudate cells. Data are mean ± SD, circles on graphs represent individual mice, n indicates the number of mice per group.

[0019] Figures 10A-F. Bone morphometric and dynamic histomorphometry effects of the deletion of Csflr in P21 Tnfrsfl la^Cre ;CsflHfl mice . Bone volume / total vlume (BV/TV,

Figure 10A), Bone length (Figure 10B), connectivity density (Conn density, Figure 10C), and Trabecular Number (Tb.N., Figure 10D) was analyzed by microCT in 21 day/old mice. Csflr (n=4) and control littermates (n=7), Tnfrsfl kT^{v sk}'^{( re} ;C.s/7/7^// (n=8) and

Tnfrsfl la^(wask)Cre+ ;CsflrT^l+(n=l), Tnfrsfl I a'"^{askl( re} ;Csflr^,u~(n=3) and control littermates (n=5). Figure 10E: Representative micrographs of calcein labeling (green) of femur of mice from the indicated genotypes and ages (n=4). Scale bars represent 50pm. Figure 10F:

Quantification of calcein labeling by fluorescence microscopy: mineralized surface/bone surface (MS/BS), mineral apposition rate (MAR), and bone formation rate/bone surface (BFR/BS) in Tnfrsfl la^(wask)Cre+ ;Csflr^¹ Csflr ⁷ (n=4) and control littermates (n=l5). Data are mean ± SD, circles on graphs represent individual mice, n indicates the number of mice per group. For statistical analysis unpaired two-tailed t-test. *p<0.05, **p<0.005,

***p<0.0005, ****p<0.000l.

[0020] Figures 11A-C. EdU labeling of bone marrow myeloid cells. Figure 11 A: Short term kinetics: EdU (20pg/g) is injected intraperitoneally to C57B16/N mice at t=0 min. Mice are sacrificed at the indicated time points and the % of EdU+ cells (blue) and the geometric MFI of EdU+ cells (red) and determined by flow cytometry, showing rapid EdU

incorporation. % of EdU+ cells plateau at ~30 min, and gMFI plateau at ~ 75 minutes.

Following a first round of cell division, -50% of monocytic cells are labeled after 8 to 12 hrs (n=3-8 mice see source data). Figure 11B: Long term kinetics: (l-240hrs) EdU is injected as in Figure 11 A and % of EdU+ monocytic cells in bone marrow (top) and blood (bottom) is determined by flow cytometry, showing that labeling of -50% of monocytic cells is observed for -2 days. Labeled cells are not detectable after 3 days. Circles represent determination from individual mice, data for each time point are pooled from 2-3 independent experiments see source data. Figure 11C: Parabiosis between Csflr^Cre ;Rosa26^LSL~YFP and

Csflr^Cre ;Rosa26^{LSL tdTomato} pairs as described in (Figure 3), paired for 1-8 weeks, and from Csflr^Cre Rosa26^LSL~YFP partners separated after 4 weeks of parabiosis and analyzed 4 weeks,

14 weeks and 24 weeks after separation. Scatter plots represent the Mean Fluorescent Intensity (MFI) of individual TRAP⁺ MGC for YFP (Y axis) and tdTomato (X axis), and histograms represent the overlaid distribution of the MFI values for YFP and tdTomato in TRAP⁺ MGC at the indicated time-points. Data are mean ± SD, circles on graphs represent individual mice, n indicates the number of mice per group.

[0021] Figures 12A-C. FACS analysis of monocyte purification, and blood / bone marrow from transferred Csflr^Cre; Csflr¹¹¹¹ mice. Figure 12A: Representative flow cytometry plots of purified bone marrow monocytes from magnetic bead based enrichment (MACS), percentage of live YFP+ monocytes is indicated. Representative flow cytometry plots from blood

(Figure 12B) and bone marrow (Figure 12C) of 14 day old mice transferred with 1E6 YFP+ monocytes on day 5,8,11, the percentage of YFP+ cells is indicated. Experiments A-C are representative of 3 independent experiments. [0022] Figure 13. Rescue of osteoclasts by monocyte transfer in Csflr^Cre; Csfld ^yT mice. High power confocal microscopy images of frozen sections from Csflr^Cre; Csflr^T mice transferred with monocytes from Csflr^Cre Rosa26^LSL~YFP and controls, stained with antibodies for YFP, TRAP substrate ELF97 and TOPRO-3 as a nuclear stain. Examples of

multinucleated Trap+, YFP+ cells (osteoclasts) are indicated with dotted lines. n=3 mice from independent litters. Numbers (#1,2,3) correspond to the mice in Figure 4D.

[0023] Figures 14A-F. Csflr deletion in Tnfrsfl la^Cre Csflr^F/F mice results in lack of tissue macrophages and osteoclasts at birth, while definitive HSC and their progeny are present in the fetal liver and blood. Figure 14A: Flow cytometry analysis of F4/80+ cells in brain (microglia) and epidermis (Langerhans cells) in El 8.5 Tnfrsfl la^Cre+ ;Csflr^F/F embryos and littermate controls. Figure 14B: H&TRAP staining of bone sections from P7

Tnfrsfl la^Cre+ ;Csflr^F/F and Tnfrsfl Ia⁽ ''-'"Csflr¹ littermate control showing absence of the bone marrow cavity. Figure 14C: Flow cytometry plot of Fetal liver at E15.5. Figure 14D LSK numbers in bone marrow of 3-4 week old Tnfrsfl la^Cre+;Csflr^F/F and littermate controls. Figure 14E: For comparison, LSK numbers in bone marrow of 3-4 week old Csflr ^A and littermate controls. Figure 14F: Blood leukocytes numbers in 4 week old

Tnfrsfl I a^{( n}' Csflr¹ mice and littermate controls. Circles represent individual mice, results from 3 independent experiments.

[0024] Figures 15A-D. Bone morphometric effects of the deletion of Csflr in P21

Tnfrsfl la^Cre+ ;Csflr^F/~ or ^F/Fmice. Bone volume / total vlume (BV/TV, Figure 15A), Bone length Figure 15B, connectivity density (Conn density, Figure 15C), and Trabecular Number (Tb.N., Figure 15D) was analyzed by microCT in 21 day/old mice of indicated genotype. Controls (ctrls) included Tnfrsflla(wask)^Cre+:Csflr^F/+ or Tnfrsfl la^(koba)Cre~ ;Csflr^F/F and Tnfrsfl la^(koba)Cre+ ;Csflr^F/+ genotypes. Circles represent 1 mouse with 3-7 mice per group. For statistical analysis unpaired two-tailed t-test. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.000l.

[0025] Figures 16A-B. EdU labeling of bone marrow myeloid cells. Figure 16A: Short term kinetics: EdET (20pg/g) is injected intraperitoneally to C57B16/N mice at t=0 min. Mice are sacrificed at the indicated time points and the % of EdU+ cells (blue) and the geometric MFI of EdU+ cells (red) and determined by flow cytometry, showing rapid EdU

incorporation. % of EdU+ cells plateau at ~30 min, and gMFI plateau at ~ 75 minutes.

Following a first round of cell division, -50% of monocytic cells are labeled after 8 to 12 hrs. Figure 16B: Long term kinetics: (l-240hrs) EdU is injected as in (A) and % of EdU+ monocytic cells in bone marrow (top) and blood (bottom) is determined by flow cytometry, showing that labeling of -50% of monocytic cells is observed for -2 days. Labeled cells are not detectable after 3 days. Circles represent determination from individual mice, data for each time point are pooled from 2-3 independent experiments.

DETAILED DESCRIPTION

[0026] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present technology are described below in various levels of detail in order to provide a substantial understanding of the present technology. The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this present technology belongs.

I. Definitions

[0027] The following terms are used herein, the definitions of which are provided for guidance.

[0028] As used herein, the singular forms“a”,“an” and“the” include plural referents unless the content clearly dictates otherwise. For example, reference to“a cell” includes a combination of two or more cells, and the like.

[0029] As used herein, the term“about” and the use of ranges in general, whether or not qualified by the term about, means that the number comprehended is not limited to the exact number set forth herein, and is intended to refer to ranges substantially within the quoted range while not departing from the scope of the invention. As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used,“about” will mean up to plus or minus 10% of the particular term.

[0030] A“chimeric nucleic acid” comprises a coding sequence or fragment thereof linked to a nucleotide sequence that is different from the nucleotide sequence with which it is associated in cells in which the coding sequence occurs naturally. [0031] As used herein, the terms“effective amount,” or“therapeutically effective amount,” and“pharmaceutically effective amount” refer to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g ., an amount which results in the prevention of a disease, condition, and/or symptom(s) thereof. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight, and tolerance to the composition drugs. It will also depend on the degree, severity, and type of disease or condition. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. In some embodiments, multiple doses are administered. Additionally or alternatively, in some embodiments, multiple therapeutic compositions or compounds are administered. In the methods described herein, compositions comprising the monocytic cells of the present technology, may be administered to a subject having one or more signs, symptoms, or risk factors of osteopetrosis, including, but not limited to stunted growth, skeletal deformity, increased likelihood of bone fracture, anemia, recurrent infections, hepatosplenomegaly, facial paralysis, abnormal cortical bone morphology, abnormal form of vertebral bodies, abnormal temperature regulation, abnormality of the ribs, abnormality of vertebral epiphysis morphology, bone pain, cranial nerve paralysis, craniosynostosis, hearing impairment, and hypocalcemia. For example, a“therapeutically effective amount” of the compositions of the present technology, includes levels at which the presence, frequency, or severity of one or more signs, symptoms, or risk factors of osteopetrosis are, at a minimum, ameliorated. In some embodiments, a therapeutically effective amount reduces or ameliorates the physiological effects of osteopetrosis, and/or the risk factors of osteopetrosis, and/or the likelihood of developing osteotpetrosis. In some embodiments, a therapeutically effective amount is achieved by multiple administrations. In some embodiments, a therapeutically effective amount is achieved with a single administration.

[0032] The term“engineered” is used herein to refer to a cell or organism that has been manipulated to be genetically altered, modified, or changed, e.g., by disruption of the genome. For example, an“engineered monocytic cell” refers to a monocytic cell that has been manipulated to be genetically altered, modified, or changed. For example, in some embodiments, an engineered monocytic cell refers to a monocytic cell that has been transduced with a lentivirus designed to express a nucleotide sequence of interest, e.g, a cDNA coding for the wild type allele of any one or more of CA2, CLCN7, CTSK, CSF1R, IKBKG, ITGB3, OSTM1, PLEKHM1 , TCIRG1 , TNFRSF11A , or TNFSF11 under a strong promoter.

[0033] “Heterologous nucleic acid” refers to a nucleic acid, DNA, or RNA, which has been introduced into a cell (or the cell’s ancestor), and which is not a copy of a sequence naturally found in the cell into which it is introduced. Such heterologous nucleic acid may comprise segments that are a copy of a sequence that is naturally found in the cell into which it has been introduced, or fragments thereof.

[0034] As used herein,“prevention,”“prevent,” or“preventing” of a disorder or condition refers to, in a statistical sample, reduction in the occurrence or recurrence of the disorder or condition in treated subjects/samples relative to an untreated controls, or refers delays the onset of one or more symptoms of the disorder or condition relative to the untreated controls.

[0035] As used herein“subject” and“patient” are used interchangeably and refer to a mammalian subject. In some embodiments,“subject” means any animal (mammalian, human, or other) patient that can be afflicted with osteopetrosis and when thus afflicted is in need of treatment. In some embodiments, the subject is a human.

[0036] “Treating,”“treat,”“treated,” or“treatment” of a disease or disorder includes: (i) inhibiting the disease or disorder, z.e., arresting its development; (ii) relieving the disease or disorder, z.e., causing its regression; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder.

[0037] It is to be appreciated that the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean“substantial,” which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.

II. General

[0038] Described herein are compositions and methods for the treatment of osteopetrosis. Taken together, the data described herein identify: (i) the developmental origin of osteoclasts, the cells that shape bone architecture; (ii) a mechanism that controls the maintenance of osteoclast function after birth; and (iii) a novel strategy to treat osteopetrosis and to modulate osteoclast activity in vivo. In particular, the data described herein demonstrate that parabiosis or transfusion of monocytic cells results in long-term gene transfer in osteoclasts in the absence of HSC chimerism and can rescue an adult-onset osteopetrotic phenotype caused by cathepsin-K deficiency. Transfusion of monocytic cells is also sufficient to rescue bone development in early-onset autosomal recessive osteopetrosis in newborn mice.

III. Osteopetrosis

[0039] Osteopetroses are a heterogeneous group of genetic disorders characterized by increased bone density due to impaired bone resorption by osteoclasts. The increased bone density places the affected individual at an increased risk for bone fracture. Normally, bone growth is a balance between osteoblasts (cells that create bone tissue) and osteoclasts (cells that destroy bone tissue). Individuals with osteopetrosis have a deficiency of osteoclasts, resulting in too little bone resorption and too much bone creation. The types of osteopetrosis are distinguished based on their pattern of inheritance: autosomal dominant, autosomal recessive, or X-linked.

[0040] The signs and symptoms of osteopetrosis may vary depending on the type of the disease, and mild forms of osteopetrosis may be asymptomatic. However, the typical signs and symptoms of osteopetrosis include: stunted growth, skeletal deformity, increased likelihood of bone fracture, anemia, recurrent infections, hepatosplenomegaly, facial paralysis, abnormal cortical bone morphology, abnormal form of vertebral bodies, abnormal temperature regulation, abnormality of the ribs, abnormality of vertebral epiphysis morphology, bone pain, cranial nerve paralysis, craniosynostosis, hearing impairment, and hypocalcemia.

[0041] The signs and symptoms of autosomal dominant osteopetrosis (ADO; also known as Albers-Schonberg disease), include multiple bone fractures, scoliosis, arthritis in the hips, and/or osteomyelitis. Autsomal recessive osteopetrosis (ARO) is often characterized by one or more of a high risk of bone fracture resulting from minor bumps or falls, pinched nerves in the head and face, impaired bone marrow function, slow growth, short stature, dental abnormalities, hepatosplenomegaly, intellectual disability, and epilepsy. Individuals diagnosed with intermediate autosomal osteopetrosis (IAO), which is a form of osteopetrosis that can have either an autosomal dominant or recessive pattern or inheritance, may be characterized by one or more of a high risk of bone fracture and anemia, calcifications in the brain, intellectual disability, and renal tubular acidosis. Individuals with the X-linked pattern of inheritance may be characterized by one or more of lymphedema, anhidrotic ectodermal dysplasia, and immunodeficiency.

[0042] Mutations in several genes have been linked to the various forms of osteopetrosis or may underlie the development of osteopetrosis. These include mutations in CA2 , CLCN7, CTSK , CSF1R , IKBKG, ITGB3, OSIMI, PLEKHM1 , TCIRG1 , TNFRSF11A (which encodes for receptor activator of NF-kB (. RANK)), and TNFSF11 (which encodes for receptor activator of NF-kB ligand (. RANKF) ). Many of the genes associated with osteopetrosis are involved in the formation, development, and function of osteoclasts.

IV. Therapeutic Applications

[0043] In some embodiments, a method for treating or preventing osteopetrosis in a subject in need thereof, comprising administering a composition comprising a therapeutically effective amount of monocytic cells from a healthy donor to the subject is provided. In some embodiments, the subject is characterized by decreased expression of one or more genes implicated in or potentially underlying the development of osteopetrosis, such as CA2 , CLCN7 , CTSK, CSF1R , IKBKG, ITGB3, OSIMI, PLEKHM1 , TCIRG1 , TNFRSF11A , and TNFSF11. In some embodiments, the subject is characterized by a cathepsin K deficiency.

[0001] [0044] In some embodiments, a method for treating or preventing osteopetrosis in a subject in need thereof, comprising administering to the subject a composition comprising a therapeutically effective amount of monocytic cells engineered to express one or more genes implicated in or potentially underlying the development of osteopetrosis, such as CA2 , CLCN7 , CTSK, CSF1R , IKBKG, ITGB3, OSIMI, PLEKHM1 , TCIRG1 ,

TNFPSF11A , and TNFSF11 is provided. In some embodiments monocytic cells used for treatment are genetically modified to correct a genetic abnormality or to improve or changed cellular functioning according to known genetic engineering protocols. In some

embodiments, a method of treating or preventing osteopetrosis in a subject comprises: (a) obtaining a sample of monocytic cells from the subject; (b) genetically correcting one or more mutations in the monocytic cells, (c) culturing the monocytic cells; and (d) providing the corrected monocytic cells to the subject. In some embodiments, a method for treating or preventing osteopetrosis comprises: (a) obtaining a sample of monocytic cells from the subject; (b) genetically engineering the cells to express a nucleotide sequence coding for the wild type allele of any one or more of CA2, CLCN7, CTSK, CSF1R, IKBKG, ITGB3 , OSTM1 , PLEKHM1 , TCIRG1 , TNFRSF11A, and TNFSF11 with a suitable transducing vector, such as a lentiviral vector; (c) culturing the engineered monocytic cells under conditions sufficient to express the nucleotide sequence; (d) removing the viral particles from the engineered monocytic cells; and (e) providing the engineered monocytic cells to the subject. In some embodiments, the transducing vector encoding the one or more of CA2 , CLCN7, CTSK , CSF1R , IKBKG, ITGB3, OSTM1, PLEKHM1 , TCIRG1 , TNFRSF11A , and TNFSF11 is a mammalian expression vector. In some embodiments, the mammalian expression vector is a lentiviral vector or transposon vector.

[0045] Subjects suffering from osteopetrosis can be identified by any or a combination of diagnostic or prognostic assays known in the art. For example, typical symptoms of osteopetrosis include, but are not limited to, stunted growth, skeletal deformity, increased likelihood of bone fracture, anemia, recurrent infections, hepatosplenomegaly, facial paralysis, abnormal cortical bone morphology, abnormal form of vertebral bodies, abnormal temperature regulation, abnormality of the ribs, abnormality of vertebral epiphysis morphology, bone pain, cranial nerve paralysis, craniosynostosis, hearing impairment, and hypocalcemia.

[0046] For therapeutic applications, a composition comprising a therapeutically effective amount of monocytic cells from a health donor and/or monocytic cells engineered to express one or more genes selected from CA2, CLCN7, CTSK , CSF1R, IKBKG, ITGB3 , OSTM1 , PLEKHM1 , TCIRG1 , TNFRSF11A, and TNFSF11 is administered to the subject. In some embodiments, the composition is administered according to any acceptable transfusion regimen. In some embodiments, the composition is administered one, two, three, four, or five times per day. In some embodiments, the composition is administered more than five times per day. Additionally or alternatively, in some embodiments, the composition is

administered every day, every other day, every third day, every fourth day, every fifth day, or every sixth day. In some embodiments, the composition is administered weekly, bi-weekly, tri-weekly, or monthly. In some embodiments, the composition is administered for a period of one, two, three, four, or five weeks. In some embodiments, the composition is

administered for six weeks or more. In some embodiments, the composition is administered for twelve weeks or more. In some embodiments, the composition is administered for a period of less than one year. In some embodiments, the composition is administered for a period of more than one year or until a desired therapeutic outcome is observed in the subject.

[0047] In some embodiments, treatment of subjects diagnosed with or suspected of having osteopetrosis with one or more compositions of the present technology ameliorates or eliminates one or more of the following symptoms of osteopetrosis: stunted growth, skeletal deformity, increased likelihood of bone fracture, anemia, recurrent infections,

hepatosplenomegaly, facial paralysis, abnormal cortical bone morphology, abnormal form of vertebral bodies, abnormal temperature regulation, abnormality of the ribs, abnormality of vertebral epiphysis morphology, bone pain, cranial nerve paralysis, craniosynostosis, hearing impairment, and hypocalcemia.

Prophylactic Methods

[0048] In one aspect, the present technology provides a method for preventing or delaying the onset of osteopetrosis or one or more symptoms of osteopetrosis in a subject at risk of having or developing osteopetrosis. In prophylactic applications, compositions of the present technology are administered to a subject susceptible to, or otherwise at risk of for

osteopetrosis in an amount sufficient to eliminate or reduce the risk, or delay the onset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.

[0049] Administration of a prophylactic compositions can occur prior to the manifestation of symptoms characteristic of the disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression.

EXAMPLES

[0050] The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. Materials and Methods

[0051] Csflr^lCre , Csflr^MenCreMer , ( 'sflr^llox were kindly provided by Dr Jeffrey Pollard, Csflr from Richard Stanley, Flt3^Cre were kindly provided by Dr Thomas Boehm, Myb^+/ mice were kindly provided by Dr John Frampton, and Vav^Cre were kindly provided from Dr Thomas Graf. Tnfrsfl 1 a^,lox mice were kindly provided by JM Penninger, Tnfrsfl ia^Koba/Cre mice were kindly provided by Dr Yasuhiro Kobayashi, Ctsk^tml(cre)Ska mice were kindly provided by Dr. Ostrowsky (MSKCC) and Tnfrsfl ]_a ^Wask/Cre mice were genereated in the Waskow Lab. Rosa.26-CreERⁿ ( R26-CreERⁿ ) were kindly provided from Drs. Pierre Chambon and Anton Bems. Rosa26^LSL~YFP (stock number: 006148) and p₀sa26^LSL~tdTomato (stock number: 007908) reporter mice were purchased from The Jackson Laboratory.

[0052] Animal procedures. Mice were bred and kept under specific pathogen conditions in separated ventilated cages in the animal facility of MSKCC and the Medical Theoretical Center of the TU Dresden. All experiments with osteopetrotic mice that lack teeth were performed with mice maximal 4 weeks of age that were kept with the lactating mother or provided with DietGel 76A (Clear H²0, 72-07-5022) to avoid secondary effects from malnutrition. Experiments were performed in adherence to the Institutional Review Board (IACUC 15-04-006) from MSKCC and Landesdirektion Dresden and were in compliance with relevant ethical regulations. Mice greater than 7 days old were sacrificed by cervical dislocation (TU Dresden), CO2 asphyxiation or anesthesia (MSKCC). To harvest embryos, pregnant females were sacrificed and embryos were collected by postmortem cesarean from the uterus and exsanguinated through decapitation in cold PBS (Fisher, 14190).

[0053] Genotyping: PCR genotyping was performed according to protocols described previously and indicated in Table 1. The investigators were not blinded to allocation during experiments and outcome assessment.

Table 1.

[0054] Fate mapping with tamoxifen inducible Cre models. For timing of embryonic development, mice were crossed at night, the following day a positive vaginal plug was considered as 0.5 days post-coitum (dpc), as previously described. Embryos were harvested as indicated below. For postnatal time point, pregnant females were monitored for the date of delivery, caesarean sections were carried out at term and neonates were fostered using lactating CD-l females.

[0055] Csflr^{U n( reUer} female mice were crossed with male Rosa26^{LSL~YFP/LSL~YFP} mice. Cre recombination in Csflr^MenCreMer ;Rosa26^LSL~YFP embryos at embryonic day E8.5 was induced with a single dose of 4-hydroxytamoxifen (4-OHT) injected intraperitoneally in pregnant mothers at a dose of 75pg/g of body weight supplemented with 37.5 pg/g of progesterone as previously described. Cre-mediated recombination in Rosa.26-CreERⁿ tamoxifen (TAM) was introduced to pregnant mother by a single TAM gavage (5mg) at El 0.5, supplemented with progesterone (37.5pg/g body weight resolved in Sunflower seed oil, Sigma-Aldrich) was injected i.p. directly after gavage. To analyze newborn or 3 weeks old mice, caesarean sections were carried out at term and neonates were fostered using lactating CD-l females.

[0056] Parabiosis. For cellular complementation female Csflr^lCre mice were crossed to male Rosa26^LSL~YFP/YFP or p_osa26^{LSL~tdTomato/tdTomato} mice. Csflr^lCre ;Rosa26^LSL~YFP and

Csflr^,Cre ;Rosa26^LSL~tdTomato females were used for parabiosis. For rescue of CathepsinK activity 4 week old female Ctsk^Cre/Cre and control littermate mice were used for parabiosis. Parabionts were kept on Sulfamethoxazole/Trimethoprim (Sulfatrim) diet for up to 8 weeks. Ex-parabionts were separated after 4 weeks for cellular complementation and 6 weeks for rescue of CathepsinK activity.

[0057] Surgical procedure, pre-operative procedure: weight-matched female partner mice for parabiosis were caged together few days before surgery. One day before the surgery the fur from lateral sides of mice was carefully removed with a trimmer followed by depilatory cream (for 3 minutes) at the site of surgery, excess of fur was removed with a moist gauze pad. Left side partner is shaved on the right side and vice versa. This procedure was performed under Isofluorane inhalation anesthesia. Mice were fed with food supplemented with Sulfatrim ad libitum one day prior to surgery.

[0058] Surgery: Mice were anesthetized intraperitoneally with l50mg/kg of ketamine and l5mg/kg of xylazine. Sterile eye lubricant (Paralube Vet Ointment, 17033-211-38) was applied to both eyes to prevent corneal drying during surgery. Following confirmation that a suitable anesthetic plane (no response to stimulation) has been attained, mice were placed in a supine position on a surgical tray with heat support provided by a heating pad. The surgical site was cleaned 3 times with cotton swabs soaked in povidone-iodine (Betadine) then with 70% ethanol. Before surgery a volume of 0.2ml of anesthetic agent bupivacaine (Marcaine 0.25-0.5% solution) was applied locally. Surgery was performed by a longitudinal skin incision on the lateral side of mice, approximately 0.5cm above the elbow to 0.5cm below the knee joint. Mice were laid side-by-side in close contact and the ligaments of the two knees and elbows were sutured together using monofilament non-absorbable suture. Then, the skin incisions was closed by apposing and clipping skin to skin of the pair with 9 mm wound clips.

[0059] Post-operative procedure: immediately after surgery mice were injected

subcutaneously with 2 mg/kg of meloxicam and 0.5mg/kg of buprenorphine and for a maximum of 48 hours postoperatively. Mice are provided with Sulfatrim and Hydrogel (Clear H2O, 70-01-5022) in medicups. Wound clips were removed 14 days after surgery under Isofluorane anesthesia.

[0060] Parabiosis separation procedure: Mice were anesthetized and prepared for surgery as indicated above. Mice were separated at the site of parabiosis junction. Using scissors, the skin joining both mice is cut longitudinally. The sutures around the elbows and knees are cut and removed. The resulting wound is closed with 9 mm wound clips. Mice were injected subcutaneously with 2 mg/kg of meloxicam and 0.5mg/kg of buprenorphine and for a maximum of 48 hours postoperatively. Wound clips were removed 14 days after surgery.

[0061] Analysis: Bones were prepared for histology on frozen sections as detailed below, and stained with antibodies to detect fluorescent proteins: anti-GFP biotin, anti-RFP

(Abeam), fluorescent TRAP staining and TO-PRO-3 as a nuclear stain. Histological sections of l5pm thickness were scanned by confocal microscopy at l.5pm Z stacks. Sections were quantified for the number of TRAP⁺ multinuclear cells (more than 3 nuclei per cell) and their YFP and Tomato expression using Imaris in 3D view and individual Z stacks. Pictures of the region of interest (area=2mm²) were then generated in Tiff format and analyzed by ImageJ software using ROI manager to calculate the mean fluorescence intensity (MFI) of YFP and Tomato for individual osteoclasts. For rescue of CathepsinK activity, dissected femurs were fixed in 10% neutral buffered formalin for 24 hours. Undecalcified bones were embedded in methyl methacrylate resin, and 7-pm sections were prepared on a rotation microtome. For mineralized bone volume over the total volume % (B V/TV%), sections were de-plastified and stained with von Kossa reagent (1% Silver nitrate/ Sodium formamide/ 5% Sodium thiosulfate) counterstained with Van Gieson solution.

[0062] EdU pulse labeling. 12 week old C57BL/6N mice (Charles River) were injected intraperitoneally with 25 pg/g of a 2.5mg/mL solution of EdU prepared extemporaneously (Fisher Cl 0420, Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit). Blood, bone marrow, and bone samples were collected at 60hrs and 72hrs post injection and bone were prepared for histology of frozen sections as indicated above for adult mice.

[0063] Histology of frozen sections. Frozen sections were cut at l5pm thickness using cryofilm and stained overnight with rat anti-Tubulin (Abeam, ab6l60, 1 :200) and with secondary goat anti-rat Alexa Fluor 555 (ThermoFisher Scientific A21430), as described below. Sections were washed 3 times with PBS and stained for EdU using Click-iT EdU Alexa Fluor 488 Imaging Assay Kit (Thermo Fisher Scientific, C10337). Click-it reaction: 357.5pL of IX Click-it reaction buffer, 40pL of CuS0₄ solution (lOOmM), 2.5pL of Alexa Fluor 488 azide solution and 100 pL of Reaction buffer additive for 500pL. Sections are incubated for 45 minutes at room temperature with lOOpL of Click-iT EdU reaction buffer then washed with PBS and stain for fluorescent TRAP (ELF 97) and TO-PRO-3 (nuclear stain). Mounting media was 75% glycerol in PBS.

[0064] Microscopy. Images were acquired using an inverted Zeiss LSM880 laser scanning confocal microscope. Histological sections of 15pm thickness were tile-scanned at l.Opm Z stacks.

[0065] Analysis. Sections were analyzed using Imaris in 3D view and individual Z stacks to quantify the percentage of EdU⁺ TRAP⁺ multinuclear cells and EdU⁺ nuclei per individual osteoclasts.

[0066] Adoptive transfers. Bone marrow monocytes were isolated from 12-16 weeks old donor. In fate-mapping experiments lxlO⁶ total cells from Csflr^,Cre ;Rosa26^{LSL tdTomato} mice were transferred at day 0, day 3 and day 6 by retro-orbital injections into recipient

Csflr^,Cre ;Rosa26^{LSL YFP} age and gender matched mice. Mice were sacrificed at day 11 and day 60 after the first transfer. For rescue of osteoclast activity lxlO⁶ total bone marrow monocytes from Csflr^lCre ;Rosa26^LSL~YFP or CsflriCre ;Rosa26^{LSL tdTomato} were transferred intra-peritoneal at p5, p8, and pl 1. Mice were sacrificed at pl4 5 days after the last transfer. Bone marrow and blood was analyzed for the percentage of chimerism. To enrich Ly6C⁺ cells the Monocyte Isolation Kit (BM) for mouse (MACS Miltenyi Biotec, 130-100-629) was used as indicated by the manufacturer. Cell numbers of Ly6C⁺ cells were calculated by determining the cell number / ml using a Neubauer chamber in combination with a staining for Ly6C analyzed by flow cytometry. Bone samples were dissected and prepared for frozen sections as described above and stained for anti-GFP, anti-RFP, fluorescent TRAP and TO- PRO-3. The percentages of YFP⁺ or Tomato⁺ multinuclear cells was quantified in femurs of recipients.

[0067] Preparation and analysis of bone for histology of paraffin embedded samples. Bone samples were fixed in 4% formaldehyde (Fisher, 28908) in PBS for 1 day (embryo) or 3 days (post-natal mice) at 4°C then washed 3 times with PBS and decalcified (for mice older than P7) in a 14% EDTA pH7.1 solution at 4°C for 5 days to 15 days, washed 3 times with PBS and dehydrated in 70% ethanol for 1 day and processed for paraffin sections. Longitudinal sections of femurs were cut at 5 pm thickness using a Leica RM2265 paraffin microtome then place in Superfrost microscope slides let dry for 48 hours and heated in a dry incubator at 65°C for 1 hour, dewaxed and stained for TRAP and hematoxylin. [0068] Tartrate Resistant Acid Phosphatase (TRAP) staining protocol . To stain for TRAP, slides were placed in coplin jars and incubated in a 1% (v/v) Naphtol -Ether substrate solution in basic stock solution for lhr at 37°C followed by incubation in a solution containing 2% (v/v) sodium nitrate solution and 2% (v/v) basic fuchsin solution in basic stock solution for 20 mins at 37°C. Slides are rinse in 3 changes of water then stained with hematoxylin solution (Sigma, GHS332) diluted 1 :4 in water for 2 minute, washed 3 times with water then dehydrated and mounted in Entellan (Millipore, 107960). Solutions for TRAP staining:. Basic stock solution: 0.92% (w/v) anhydrous sodium acetate (Sigma, S8750), 1.14% (w/'v) dibasic dihydrate sodium tartrate (Sigma, S4797) and 0.28% (v/v) glacial acetic acid (Sigma,

537020) in distilled water, pH was adjusted between 4.7-5.0 with 5M sodium hydroxide

(Fisher, S318-1). Napthol-Elher substrate solution: 2% (w/v) Napthol AS-B1 Phosphate (Sigma, 70482) in 2-Ethoxy ethanol (Sigma, 256374). Sodium nitrate solution: 4% (w/v) Sodium nitrate (Sigma, 237213) in water. Basic fuchsin solution: 5% (w/v) basic fuchsin dye (Sigma, 857343) in 2N HCL (Fisher, A144-500).

[0069] Individual images from histological sections of 5pm thickness were acquired using a Zeiss Axio Lab.Al light microscope with a N-Achroplan 2.5X/0.07 M27 (420920-9901) or a N-Achroplan 20X/0.45 M27 (420950-9901) objective. Pictures were taken in ZEN lite software and exported as tiff files. Panoramic images were created with the photo-merge tool in Adobe Photoshop CS6. Pictures of mice were acquired with a dissecting microscope Leica M80 equipped with a Leica IC80 HD camera at 1.0X magnification. The region of interest analyzed was the methaphyseal trabecular bone 2 mm below the growth plate. The numbers of TRAP⁺ multinuclear cells (more than 3 nuclei per cell), associated to bone tissue, were quantified in ImageJ using the Cell Counter plugin. Numerical values were plotted using GraphPad Prism. For Static and dynamic histomorphometry. Young and aged mice were injected (i.p.) twice with 15 mg/kg body weight Calcein (Sigma) dissolved in 1.4%

NaHC03/PBS 2 and 3 days apart, respectively. Mice were sacrificed 2 days after the last calcein injection. Femora and tibiae were fixed in 4% PBS-buffered paraformaldehyde and dehydrated in an ascending ethanol series. Subsequently, bones were embedded in methacrylate and cut into 7 pm sections to assess the fluorescent calcein labels. Unstained sections were analyzed using fluorescence microscopy to determine the mineralized surface/bone surface (MS/BS), the mineral apposition rate (MAR), and the bone formation rate/bone surface (BFR/BS).To determine numbers of osteoclasts, bones were decalcified for one week using Osteosoft (Merck), dehydrated, and embedded into paraffin. Tartrate- resistant acid phosphatase staining was used to assess the osteoclast surface per bone surface (Oc.S/BS) and number of osteoclasts per bone surface (N.Oc/BS). Bone sections were analyzed using the Osteomeasure software (Osteometries, USA) following international standards.

[0070] Preparation of and analysis of bones for immunofluorescence on frozen sections. Samples were prepared as above and after decalcification and washing were soaked in 30% sucrose in PBS at 4°C for 1-2 days. Tissue samples were placed in disposable histology plastic molds and embedded in FSC22 Frozen Section Compound Clear (Leica, 3801480) and placed on a flat surface of dry ice to let freeze.

[0071] Immunofluorescence. Bones were cut at 15 pm thickness using a Cryostat Leica CM3050S with high profile microtome blades (Leica Surgipath DB80 HS) and cryofilm (Section Lab Inc.,) and let to dry for 48 hours at 4°C. Before staining with antibodies, sections were let to equilibrate at room temperature for 30 minutes, rehydrated with PBS (Fisher, 14190) 3 times for 5 min at room temperature. Sections were incubated with blocking buffer containing 0.25% BSA (Fisher BP 1600), 10% normal goat serum (Life Technologies, PCN 5000) and 0.3% triton (Sigma, T8787) in PBS for lhr at room temperature. Sections were washed 2 times with PBS for 5 minutes. Sections stained with anti-GFP biotin antibody were first incubated with Biotin/Streptavidin blocking kit (Vector laboratories, SP2002). Streptavidin blocking solution is prepared by adding 4 drops of streptavidin solution to lmL of PBS/0.25% BSA, samples were incubated for 15 min, then washed once with PBS for 5 min. Biotin blocking solution is prepared by adding 4 drops of biotin solution to lmL of PBS/0.25% BSA, samples were incubated for 15 min then washed once with PBS for 5 min. Primary and secondary antibodies used are listed in Table 2. Sections were also stained with fluorescent TRAP and nuclear stain.

Table 2.

Fate-mapping models: FU3^Cre;Rosa26^{LSL YFP}, Cfslr^Cre;Rosa26^{LSL YFP} Csfl r^MeriCreMer;Rosa26^{LSL~ YFP} and Tnfrsflla^Cre;Rosa26^{LSL YFP}

Parabiosis

Bone marrow monocyte

transfusions and transplants

EdU

staining*

*After antibody staining sections were stained with the Click-it EdU

Alexa Fluor 488 Imaging Assay kit, as described in methods.

[0072] Fluorescent TRAP staining. Sections were prepared for fluorescent TRAP by incubating with TRAP incubation solution (112 mM sodium acetate, 76 mM sodium tartate, and 11 mM sodium nitrite, pH 4.1-4.3) at room temperature for 10 minutes. Buffer was removed and incubated with ELF97 substrate (Molecular Probes E6589, 2mM) at a concentration of 125mM in TRAP incubation solution for 15 min under ETV light and washed 2 times with PBS for 5 minutes. Nuclear stain used was TO-PRO-3 Iodide (Fisher T3605) 1 :4000 in PBS for 5 minutes. Mounting media was 75% glycerol in PBS. Images were acquired using an inverted Zeiss LSM880 laser scanning confocal microscope with Argon- ion 488nm, Diode 405-30nm, DPSS 56l-l0nm, HeNe 633nm laser lines and Plan- Apochromat 40X/1.4 N.A. DIC (UV) VIS-IR oil objective. Histological sections of l5pm thickness were tile-scanned at l .5pm Z-stacks in ZEN black and processed using ZEN lite. Sections were analyzed using Imaris in 3D view and individual Z stacks to quantify TRAP⁺ multinuclear cells and YFP and Tomato labeling. Pictures of the region of interest were generated in tiff format and analyzed by ImageJ software using ROI manager to calculate the mean fluorescence intensity (MFI) of individual osteoclasts. Numerical values were plotted using GraphPad Prism.

[0073] Animal imaging by computed tomography. NanoSPECT/CT: Mice were

anesthetized under Isofluorane anesthesia and placed on an imaging table containing an animal bed equipped with a nosecone for gas inhalation and body temperature stabilization. For 3 week old mice a mouse bed was used and for mice 8 weeks and older a rat bed was used. Whole-body imaging of mice was acquired using a NanoSPECT/CT scanner (Mediso) for non-invasive and longitudinal monitoring of the 3D skeletal structure. Each CT scan averaged 15 minutes and was acquired with an exposure time of 1, 000ms and 240 projections set at a pitch of 1 degree. The tube energy of the X-ray was 55KVp and 145mA. The in-plane voxel size was medium generating a voxel size of 147pnr\ Reconstructed images were analyzed using In Vivo Scope 2.0 (Bioscan, Inc.) software.

[0074] microCT. For tri-dimensional X-ray imaging by micro computed tomography, mice were sacrificed and bones placed in 70% ethanol until scanning. Bone microarchitecture was analyzed using the vivaCT40 (Scanco Medical, Switzerland). Entire femora or humeri were imaged at a resolution of 10.5pm (1 slice) with an X-ray energy of 70 kVp, 114 mA, and an integration time of 200ms. The machine was routinely calibrated using hydroxyapatite phantoms for density and geometry. Trabecular bone in femora or humeri from old mice was assessed in the metaphysis 20 slices below the growth plate using 150 slices. The trabecular region within the cortical bones (P21 mice) was determined in the femoral midshaft (100 slices up, 100 slices down). Pre-defmed scripts from Scanco were used for the evaluation.

[0075] Preparation of tissues and staining for Flow cytometry. Yolk sac from E10.5 embryos was digested for 60 min at 37°C in PBS containing 5% FCS, Collagenase Type 4 (Worthington, final 4.2 U/ml) and DNAsel (Sigma- Aldrich, final 100 pg/ml). The digestion reaction was stopped by incubation with 12.5 mM EDTA. Fetal liver was gently dissociated between the frosted ends of glass slides, and was then digested for 30 minutes using the same digestion enzyme mix as yolk sac. Blood was collected from anesthetized mice by retro- orbital venous sinus bleeding or cardiac puncture using a lmL syringe and a 26G needle rinsed with lOOmM EDTA (sigma E4884). The collected blood was lysed with 3 mL of red blood cell lysis buffer (l55mM NFECl sigma A9434, lOmM NaFlCCh sigma S5761 and O. lmM EDTA sigma E4884) for 5 mins, washed with lOmL of FACS buffer (PBS, 0.5%

BSA and 2mM EDTA) and centrifuged at 320g for 7 minutes at 4°C. All mice were perfused with lOml of PBS after blood withdrawal. Bone marrow cells were harvested by crushing or flushing femurs and tibias using a syringe and a 26G needle with lOmL of PBS / 5% FCS or RPMI supplemented with 10% FBS. Bone marrow was dissociated by gently pipetting up and down with a lmL or lOmL pipette. Spleens were gently dispersed between frosted slides and digested for 30 min at 37 °C in PBS with 5% FCS containing Collagenase Type 4 (Worthington) at a final concentration of 4.2 El/ml and 100 pg/ml DNAsel (Sigma- Aldrich). The reaction was stopped by incubation with 12.5 mM EDTA. Peritoneal cells were harvested by flushing the peritoneal cavity with lOmL PBS/5% FCS heated to 37°C. Adult brain and liver were dissected, cut into small fragments, and incubated at 37°C for 30 min in enzyme mix consisting of PBS with lmg/ml collagenase D (Sigma, 11088882001), 100 U/ml DNase I (Sigma, DN25), 2.4mg/ml of dispase (Fisher, 17105-041) and 3% FBS (ThermoFisher Scientific 10438026) or PBS containing 4.2 U/ml Collagenase type 4 (Worthington), 100 pg/ml DNAsel, 2.4mg/ml of Dispase (Gibco) and 3% FCS at 37°C for 30 min. After enzyme digestion all tissues were further dissociated by mechanical disruption using lOOpm cell strainers (Falcon, 352360) and a 3ml syringe plunger in 6 well plates containing 4mL or 5mL of cold FACS buffer or PBS / 5% FCS. Single cell suspensions were transferred to 5mL FACS tubes and pelleted by centrifugation at 320g for 7 minutes at 4°C. The cell pellets were resuspended in FACS buffer containing purified anti-mouse CD16/32 antibody (1 : 100, Biolegend 101301), 5% normal mouse (Fisher, 015-000-120), 5% normal rat (Fisher 012-000-120) and 5% normal rabbit serum (Fisher, 011-000-120) and incubated for 10 minutes on ice or directly stained using blocking antibodies in the staining mixtures. Samples were immunostained with fluorochrome-conjugated antibodies for 30 minutes on ice, and analyzed by flow cytometry using an LSRFortessa or an LSR II (BD-Bioscience). Full list of antibodies for flow cytometry is provided in Table 3. Each sample was stained with Hoechst (ThermoFisher Scientific, Hoechst 33258, 1 pg/ml) or DAPI (Applichem, l pg/25ml) moments prior to flow cytometry acquisition.

Table 3.

BM: Bone marrow; SP: Spleen; BL: Blood; Y: Yolk sac; FL: Fetal Liver; LV: Liver; LN: Lung; BR: Brain; P: PEC: E: Epidermis; K: Kidney.

[0076] All analysis was conducted using FlowJo (Tree Star). In all tissues single live cells were gated by exclusion of dead cells labeled positive by Hoechst or DAPI, side scatter (SSC-A) and forward scatter (FSC-A) and doublet exclusion using forward scatter width (FSC-W) against (FSC-A), as previously described. In order to calculate cell numbers per organ or per gram of tissue, organs were weighted, cell suspensions were prepared from a weighted amount (20 to 500mg) of tissue, and the number of cells per gram of tissue was determined using a cell counter (GUAVA easyCyte HT).

[0077] Statistical analysis and reproducibility. Data are shown as mean with individual values per mouse represented as circles, unless stated otherwise. Statistical significance was analyzed with GraphPad Prism using unpaired t-tests and two-way ANOVA with Tukey’s multiple comparisons test as indicated in the figure legends. Significance was considered at P value (P) *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The n value represents biological replicates. Experiments were repeated to ensure reproducibility of the

observations. Equal variance was assumed for cell-counting experiments. No statistical methods were used to predetermine sample size. Example 1 : Osteoclast origins and maintenance

[0078] In vitro , osteoclasts arise by fusion of HSC-derived precursors and require expression of Csflr and Tnfrsfl la (Rank). To probe the origin of osteoclasts in vivo ,

Csflr^Cre; Csflr¹¹¹¹; and Csfif ^rc; Tnfrsfl la¹¹¹¹ mice were generated. These mice presented with an osteopetrotic phenotype similar to Csfl (op/op), Csflr , and Tnfrsfl la mutants and characterized in young mice by lack of teeth eruption, skull and skeletal deformities with shortness of long bones, increased bone density, and lack of osteoclasts and hematopoietic cells (Figure 1A; Figure 5B). To confirm that osteoclast differentiation requires expression of Tnfrsfl la and Csflr in HSC-derived precursors, Flt3^Cre ; Tnfrsfl lcfi^ mice, 1Ή3^{( n‘};CsfIr^{/! /!} mice, and Vav^Cre; Csfir^/lJlm\ce were generated. Surprisingly, all young mice had normal teeth (Figure IB) and bone morphology (Figure 6A-C), normal bone marrow cellularity (Figure 1C), and normal osteoclast numbers (Figure ID) in comparison to control littermates.

However, 1Ίΐ3^{( r} ; Tnfrsfl I a¹¹¹¹ mice, Flt3^Cre ;CsflrFfl mice, and Vav^Cre; CsflrFA mice lost their osteoclasts over time (Figure ID), and by 22-60 weeks of age had increased trabecular bone density (Figure IE), decreased hematopoietic cell numbers in the long bones (Figure 1C; Figure 6D-G) and tri-dimensional X-ray imaging by micro computed tomography (micro- CT) confirmed increased bone mass while bone formation measured by calcein incorporation was similar to control(Figure IF, Figure 7A-E).

[0079] Mice lacking Csflr expression in Flt3^Cre-ex pressing progenitors presented with a similar phenotype: Flt3^Cre CsflrTl¹ mice have normal teeth and bone morphology at 4 week of age (Figure IB, Figure 6C), but osteoclast number are decreased over time and bone mass is increased at 22 weeks of age (Figure 1D-E, Figure 7A-E). In a complementary approach to delete Csflr in HSC-derived progenitors, Vav^Cre ;CsflrFfl mice were generated. Again,

Vav^Cre CsflrFfl mice were not osteopetrotic at birth, had normal teeth (Figure IB), and hematopoietic cell numbers in long bones at 4 week of age (Figure 1C). However, they developed a late-onset osteopetrosis similar to Flt3^Cre ;Tnfrsfl lcfT and Flt3^Cre CsflrH¹ mice: at ~60 weeks of age Vav^{( n}';Csf I r^{/! 11} mice had decreased osteoclasts and hematopoietic cell numbers in long bones (Figure 1C-D), enlarged trabecular bone (Figure IE) and increased bone mass (Figure IF, Figure 7A-E). These data suggested that osteoclast development, tooth eruption and the development of bone and the bone marrow cavity requires precursors that express Csflr, but not Flt3^Cre or Vav^Cre although postnatal contribution of Flt3^Cre / Vav^Cre expressing cells is important for optimal osteoclast function in adults and aging mice. [0080] Fetal expression of Csflr^Cre and Flt3^Cre was analyzed using a fluorescent reporter in Csflr^Cre; Rosa26^{LSL YFP} and Flt3^Cre; Rosa26^{LSL YFP} mice (Figure 1 G-I). TRAP⁺

multinucleated cells that appear at embryonic day (E)l5 in ossification centers are labeled with YFP in Csflr^Cre;Rosa26^{LSL YFP} mice and osteoclasts remain YFP positive throughout life (Figure 1 G,I; Figure 8A-E), but gain expression of YFP after birth in Flt3^Cre ;Rosa26^{LSL YFP} mice despite colonization of the fetal bone marrow by Flt3^Cre+ YFP⁺ hematopoietic cells (Figure 1 H; Figure 8A-E). These data suggested that although postnatal contribution of HSC-derived cells is important for optimal osteoclast maintenance and function in adults and aging mice, osteoclast development, tooth eruption, the development of bone, and the bone marrow cavity requires precursors independent from the HSC lineage, possibly from the embryonic EMP lineage of resident macrophages because Csflr^Cre mice allow deletion of target genes in both the embryonic EMP lineage and in the HSC lineage, while Flt3^Cre and Vav^Cre (Figure 9A-D) are not expressed in the EMP lineage.

[0081] In support of this hypothesis, it was found that TRAP⁺ multinucleated cells develop in ~E 15 ossification centers from A/) i-deficient embryo, which lack HSC but still support the development of EMP-derived macrophages (Figure 2A, B). In addition, TRAP⁺

multinucleated cells are labeled with YFP in Csfh^Mer~lCre~Mer; Rosa26^LSL~YFP mice pulsed at E8.5 with a single dose of hydroxytamoxifen (4-OHT), which labels EMP but not HSC

(Figure 2C, D; Figure 8A-E). Altogether, these results indicate that fetal osteoclasts arise from EMP in ossification centers.

[0082] Whether EMP are required for bone development was investigated. Tnfrsflla is expressed by osteoclasts, but its expression is also a hallmark of EMP-derived pMacs that colonize the developing embryo. In two independent lines oiTnfrsfl la^Cre knock-in mice, Cre- mediated expression of a Rosa26^LSL~YFP fluorescent reporter is achieved with high efficiency in fetal macrophages but with low efficiency or not at all in HSC and their progeny in blood and tissues (Figure 9A-D). It was therefore hypothesized that conditional deletion of Csflr in Tnfrsflla^Cre would recapitulate macrophage deficiency observed in C.s/7/ -deficient mice, while leaving the HSC lineage unaffected. To test osteoclast and bone development in this model, two independent lines of Tnfrsfl la^Cre ;CsflrFA mice were generated (Figure 2E-J). Tnfrsfl la^Cre ;CsflrFfl lack tissue macrophages such as brain microglia and epidermal

Langerhans cells at birth while development of HSCs and blood cells was preserved (Figure 6A-N). They present with a severe osteopetrotic phenotype including the lack of tooth eruption (Figure 2E), misshaped skulls and short long bones (Figure 2F-H; Figure 5A-G) with few osteoclasts (Figure 21), increased bone density and initially lack a bone marrow cavity (Figure 2F; Figure 6A-N). In contrast to C.s/// -deficient mice, however, osteoclasts and hematopoietic cells progressively colonized the long bones of Tnfrsfl la^Cre iCsflr^ mice during the first month of life (Figure 21; Figure 5A-G; Figure 6A-N) although the mice remained toothless and skull and long bone deformity persisted (Figure 2E-H; Figure 5A- G). Calcein incorporation was similar in Tnfrsl Id ^r, ;CsfIr¹¹¹¹ mice and their littermate controls (Figure 10A-F). In a complementary approach ablation of Csflr expression in E10.5 embryos using a single dose of tamoxifen in Rosa26-creER^T2+ ,-Csflr^ mice resulted in defective tooth eruption in 3 out of 4 pups at 21 days of age (Figure 9A-D). Altogether, this analysis supports a model in which EMP-derived embryonic osteoclasts are needed for teeth eruption, normal skull shape and optimal formation of long bones, and the timely

colonization of long bones by hematopoietic progenitors, while HSC-derived osteoclasts are important for the maintenance of bone mass after birth and later in life, although they may partially rescue bone development in the absence of EMP-derived osteoclasts in

Difrsl Id ^re;Csflr¹¹¹¹ mice.

Example 2: Therapeutic approach for the treatment of osteopetrosis

[0083] To probe the mechanisms that underlie the contribution of HSC-derived blood cells to osteoclast maintenance as well as the lifespan and dynamics of osteoclasts in vivo , time- course parabiosis experiments were performed (Figure 3A). After 4 to 8 weeks of shared blood circulation between Csflr^Cre ;Rosa26^LSL~YFP and Csflr^Cre ;Rosa26^{LSL tdTomato} parabionts, all osteoclasts, defined as TRAP⁺ multinucleated cells lining the bone surface, expressed both YFP and tdTomato within the same cells (Figure 3A, B). No other cell was found to co- express YFP and tdTomato in bones (Figure 3B). This is compatible with the presence of nuclei from both partners in individual osteoclasts. Moreover, when parabionts were separated after 4 weeks (Figure 3C), most recipient YFP+ osteoclasts retained tdTomato staining 14 weeks after separation, and two third of osteoclasts from former parabionts still expressed both YFP and tdTomato 24 weeks after separation (Figure 3C). TdTomato signal intensity per YFP osteoclast increased during the 8-week period of shared blood circulation, and decreased after separation (Figure 11A-C). Most murine osteoclasts have ~5 (3 to 7) nuclei, with a modest increase of nuclei per cell between 1 month and 6 months of age

(Figure 3E). These data therefore suggest that individual osteoclast syncytia are long lived, but acquire new nuclei one by one every 4 to 8 weeks from circulating blood cells so that it takes more than 6 months to renew all 5 nuclei from a given osteoclast.

[0084] The number and fusion-rate of HSC-derived nuclei acquired by osteoclasts in short term 5-ethynyl-2’-deoxyuridine (EdU) incorporation studies was calculated. A single intravenous pulse of EdU (20pg/g) labels mitotic nuclei, is bioavailable in the bone marrow for ~90 min, and -50% of bone marrow and blood monocytic cells are EdU⁺ for ~48hrs

(Figure 11A-C). It was observed that -1 to 2% of osteoclasts were labelled after 72 hrs, with only 1 EdU⁺ nucleus per osteoclast in 90% of positive cells (Figure 3F) suggesting acquisition by osteoclasts of single post-mitotic nuclei at a time. The proportion of osteoclasts acquiring a new nucleus can be estimated -0.5 to 2 % per day in this model, compatible with individual nuclei being replaced every - 2 months. Altogether the studies above suggested a model (Figure 3G) where: osteoclasts that control skeletal development develop in ossification centers from EPM, while their post-natal maintenance is mediated via the serial acquisition by long-lived syncytia of new nuclei from HSC-derived blood leukocytes, rather than by de novo renewal by lateral fusion or proliferation of osteoclast precursors.

[0085] A prediction from this model is that osteopetrosis due to a recessive mutation affecting osteoclasts function may be rescued or prevented through parabiosis with a wild- type partner. Parabiosis experiments between 4 week-old cathepsin K deficient mice, which develop an adult-onset form of osteopetrosis known as pycnodysostosis, and cathepsin K^+/ or cathepsin K littermates and between wild type mice as control showed a reduction of bone volume in 10 weeks old cathepsin K mice paired with cathepsin K^+/ littermates (Figure 4A), suggesting that circulating blood cells carrying a wild type cathepsin K allele are sufficient to reduce bone density. To confirm that expression of a donor-derived gene by recipient osteoclasts results from fusion with blood circulating monocytic cells, intravenous injections of Kit Ly6C⁺ cells from the bone marrow of Csflr^Cre;Rosa26^{LSL tdTomato} into Csflr^Cre;Rosa26^{LSL YFP} recipients were performed (Figure 4B; Figure 12A-C). This resulted in stable expression of tdTomato in 20-40% of osteoclasts 1 week and 8 weeks after transfusion, in the absence of other donor-derived blood cells or bone marrow progenitors (Figure 4B, C; Figure 12A-C). These results suggest that parabiosis or an appropriate regimen of transfusion can achieve expression of a donor-derived gene by recipient osteoclasts in the absence of HSC chimerism, and in addition that this effect can last for several months. [0086] Partial rescue of osteopetrosis occurs postnatally in Tr/rsfl Ia^{( r,} ;CsfIr¹¹¹¹ mice, suggesting that transfusion of monocytic cells may also be able to rescue bone development in early-onset congenital osteopetrosis in the absence of a bone marrow transplantation. Intra- peritoneal injections of Kit Ly6C⁺ monocytic cells from Csflr^Cre;Rosa26^{LSL YFP} into Csflr^Cre; Csflr^F/F neonates starting from post-natal day 5, resulted in complete or partial rescue of teeth eruption (Figure 4D) and long bone development as assessed by CT-Scan, with the development of a bone marrow cavity (Figure 4D) at day 14 in infant mice from 3 different litters (Figure 4D, Figure 13). In all mice, histology of femurs indicated the presence of numerous YFP+ TRAP+ osteoclasts lining the bone (Figure 4E, F). Histology and flow cytometry analyses showed the lack of YFP+ circulating blood cells or bone marrow progenitors (Figure 4E, G), indicating absence of HSC engraftment. These data suggest that transfusion of monocytic cells can rescue bone development in early-onset autosomal recessive osteopetrosis in infant mice, in the absence of HSC transplantation.

Summary

[0087] The results described above show that osteoclasts originating from EMP are essential for normal bone development. Moreover, it is shown that osteoclasts are long-lived in adult and their function is maintained by iterative fusion of individual HSC-derived circulating cells with existing syncytia. In the absence or deficiency of EMP-derived osteoclasts, however, their timely replacement by transfusion with monocytic cells can rescue bone development in early-onset osteopetrotic mice in the absence of bone marrow transplantation. This could contribute to the treatment of early onset osteopetrosis, since the current treatment by bone marrow transplantation. This is of potential clinical relevance because bone marrow/HSC transplantation, the standard of care in early-onset osteopetrosis in mice and human, requires irradiation or chemotherapy which carry the risk of threatening infections and is frequently performed late in patients who already suffer severe

complications and is plagued by a -48% overall survival at 6-years. In addition, the original mechanism that mediates osteoclast maintenance suggests that they represent a unique target for gene transfer by cellular therapies based on transfusion of wild type or engineered monocytic cells to modulate osteoclast activity and bone remodeling in adults. Accordingly, these results demonstrate that monocytic cells may be effective in methods for treating osteopetrosis, and to modulate osteoclast activity and bone remodeling. Example 3: Ex vivo gene therapy for the treatment of a form of osteopetrosis: pycnodvsostosis

[0088] This example demonstrates the prophetic use of monocytic cells obtained from a subject and engineered to express a gene, such as CTSK , for the treatment of osteopetrosis in the subject.

[0089] Pycnodysostosis is an autosomal recessive form of osteopetrosis, due to loss of function mutations in the cathepsin K gene (CTSK), compatible with life, and characterized by short stature, deformity of the skull maxilla and phalanges, increased density of the bones, osteosclerosis, and fragility of bone. There is no treatment for this disease as the relatively comparably milder symptoms do not qualify for bone marrow transplantation.

[0090] Subjects suspected of having or diagnosed as having cathepsin K deficiency will be selected for a gene therapy trial and will undergo GMP grade collection of peripheral blood monocytes by apheresis and/or elutriation. Purified monocytes will then be transduced with a lentivirus designed to express a cDNA coding for the wild type allele of CTSK under a strong promoter ( e.g ., CMV). Transduced monocytes will then be washed to remove contaminating viral particles and rested for 12 hours followed by reinfusion to the patient. Assuming -10⁹ monocytes can be collected, the number of cells transferred per kilogram of body weight would be >l0⁷. Following the auto-transfusion, the subject will be monitored at regular intervals for clinical and radiological signs of pycnodysostosis in order to determine the efficiency of the procedure and the frequency with which it should be repeated.

[0091] It is predicted that subjects suspected of having or diagnosed as having

pycnodysostosis and receiving therapeutically effective amounts of monocytic cells engineered to express CTSK will display reduced severity or elimination of one or more symptoms associated with pycnodysostosis. Accordingly, these results will show that transfusion of monocytic cells engineered to express the wild-type version of one or more genes implicated in osteopetrosis is useful in methods for treating subjects in need thereof for the treatment of osteopetrosis. Example 4: Ex vivo gene therapy for the treatment of a form of osteopetrosis: severe infantile autosomal recessive osteopetrosis

[0092] This example demonstrates the prophetic use of monocytic cells obtained from a subject and engineered to express a gene, such as TCIRG1, TNFRSFlla, CA2, CLCN7, OSTM1, and/or PLEKHM1, for the treatment of osteopetrosis in the subject.

[0093] Infants diagnosed with severe infantile autosomal recessive osteopetrosis with defects in TCIRG1, TNFRSFlla , CA2 , CLCN7 , OS/M I, and/or PLEKHM1 will be eligible for an early auto-transfusion of corrected monocytes (as described in Example 3), to try to prevent early developmental complications, including blindness and skeletal deformities, and increase the success of bone marrow transplantation, by restoring a bone marrow niche. The protocol will be carried out in a manner similar to that of Example 3, and monocytes will be transduced with a lentivirus designed to express a cDNA coding for the wild type allele of the deficient gene under a strong promoter, with a similar number of transferred cells per kg of weight. Following the auto-transfusion, subjects will be monitored at regular intervals for clinical and radiological signs, and possibly bone biopsy in order to determine the efficiency of the procedure. In addition, collection of monocytes and hematopoietic stem

cells/progenitors at the same time would allow to proceed with the auto transfusion of transduced monocytes, while stem cells would undergo gene editing in view of auto transplantation and long term genetic rescue.

[0094] It is predicted that subjects suspected of having or diagnosed as having severe infantile autosomal recessive osteopetrosis and receiving therapeutically effective amounts of monocytic cells engineered to express TCIRG1 , TNFRSFlla , CA2, CLCN7 , OSTM1, and/or PLEKHM1 will display reduced severity or elimination of one or more symptoms associated with severe infantile autosomal recessive osteopetrosis. Accordingly, these results will show that transfusion of monocytic cells engineered to express the wild-type version of one or more genes implicated in osteopetrosis is useful in methods for treating subjects in need thereof for the treatment of osteopetrosis. REFERENCES

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EQUIVALENTS

[0095] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present technology is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0096] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0097] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as“up to,”“at least,”“greater than,”“less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth. [0098] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0099] Other embodiments are set forth within the following claims.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for treating or preventing osteopetrosis in a subject in need thereof, the method comprising administering a composition comprising a therapeutically effective amount of monocytic cells from a healthy donor to the subject.

2. The method of claim 1, wherein the subject is characterized by decreased expression of one or more of CA2, CLCN7 , CTSK, CSF1R , IKBKG, ITGB3, OSTM1, PLEKHM1 , TCIRG1 , TNFRSF11A, and TNFSF11, as compared to the monocytic cells of the donor.

3. The method of claim 1, wherein the osteopetrosis comprises one or more of stunted growth, skeletal deformity, increased likelihood of bone fracture, anemia, recurrent infections, hepatosplenomegaly, facial paralysis, abnormal cortical bone morphology, abnormal form of vertebral bodies, abnormal temperature regulation, abnormality of the ribs, abnormality of vertebral epiphysis morphology, bone pain, cranial nerve paralysis, craniosynostosis, hearing impairment, and hypocalcemia.

4. The method of claim 1, wherein the composition is formulated for intravenous

administration by injection, infusion, or transfusion.

5. The method of claim 1, wherein the subject is a mammal.

6. The method of claim 5, wherein the mammalian subject is a human.

7. The method of claim 1, wherein the subject is characterized by a cathepsin K

deficiency.

8. A method for treating or preventing osteopetrosis in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of monocytic cells engineered to express one or more genes selected from CA2, CLCN7, CTSK, CSF1R, IKBKG, ITGB3, OSTM1 ,

PLEKHM1, TCIRG1 , TNFRSF11A , and TNFSF11.

9. The method of claim 7, wherein the osteopetrosis comprises one or more of stunted growth, skeletal deformity, increased likelihood of bone fracture, anemia, recurrent infections, hepatosplenomegaly, facial paralysis, abnormal cortical bone morphology, abnormal form of vertebral bodies, abnormal temperature regulation, abnormality of the ribs, abnormality of vertebral epiphysis morphology, bone pain, cranial nerve paralysis, craniosynostosis, hearing impairment, and hypocalcemia.

10. The method of claim 8, wherein the composition is formulated for intravenous

administration by injection, infusion, or transfusion.

11. The method of claim 8, wherein the subject is a mammal.

12. The method of claim 11, wherein the mammalian subject is a human.

13. The method of claim 8, wherein the subject is characterized by a cathepsin K

deficiency.

14. The method of claim 8, wherein the monocytic cells are obtained from the subject.

15. A donor monocytic cell line engineered to express one or more genes selected from CA2 , CLCN7 , CTSK, CSF1R , IKBKG, ITGB3, OSTM1, PLEKHM1 , TCIRG1 , TNFRSF11A , and TNFSF11 , wherein the one or more genes is operably linked to a heterologous nucleic acid to form a chimeric nucleic acid construct.

16. The donor monocytic cell line of claim 15, wherein the heterologous nucleic acid encodes a selectable marker.

17. The donor monocytic cell line of claim 16, wherein the selectable marker is a

bioluminescent protein, a fluorescent protein, a chemiluminescent protein, a xanthine- guanine phosphoribosyl transferase gene (gpt), or any combination thereof.

18. The donor monocytic cell line of claim 15, wherein the heterologous nucleic acid encodes one or more control sequences suitable for directing expression of the one or more genes in a monocytic cell.

19. The donor monocytic cell line of claim 18, wherein the one or more control sequences comprises a promoter.

20. The donor monocytic cell line of claim 15, wherein the cells comprise a vector

encoding one or more genes selected from CA2, CLCN7 , CTSK , CSF1R, IKBKG, ITGB3 , OS/MI, PLEKHM1, TCIRG1, TNFRSF11A, and TNFSF11.

The donor monocytic cell line of claim 20, wherein the vector is a mammalian expression vector, a lentiviral vector, or transposon vector.