WO2017173103A1

WO2017173103A1 - Regulation of gene expression by modulating primary cilia length

Info

Publication number: WO2017173103A1
Application number: PCT/US2017/025063
Authority: WO
Inventors: Christopher Jacobs JACOBS; Milos SPASIC
Original assignee: Columbia University in the City of New York
Current assignee: Columbia University in the City of New York
Priority date: 2016-03-30
Filing date: 2017-03-30
Publication date: 2017-10-05
Anticipated expiration: 2018-09-30
Also published as: US20190015423A1

Abstract

The presently disclosed subject matter relates to methods of regulating gene expression in a cell by modulating the length of a primary cilia of a cell, wherein such modulation can modulate the mechanosensitivity of the cell. The presently disclosed subject matter also provides for methods of treating ciliopathies and osteoporosis in a subject by increasing the length of a primary cilia of a cell in a subject.

Description

REGULATION OF GENE EXPRESSION BY MODULATING PRIMARY

CILIA LENGTH

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Application Serial No.

62/315,545 filed on March 30, 2016, which is incorporated in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The subject matter of the instant disclosure was made with government support under Grant No. RO1-AR062177 awarded by National Institute of Arthritis and Musculoskeletal and Skin Diseases/National Institute of Health (NIAMS/NIH). The government has certain rights in the invention.

FIELD OF INVENTION

The presently disclosed subject matter relates to methods of regulating gene expression in a cell by modulating the length of primary cilia of the cell. The presently disclosed subject matter also provides for methods of treating ciliopathies and osteoporosis.

SEQUENCE LISTING

The specification further incorporates by reference the Sequence Listing submitted herewith via EFS on March 30, 2017. Pursuant to 37 C.F.R. § 1.52(e)(5), the Sequence Listing text file, identified as seqlisting03292017.txt, is 656 bytes and was created on March 29, 2017. The Sequence Listing, electronically filed herewith, does not extend beyond the scope of the specification and thus does not contain new matter.

BACKGROUND

Mechanotransduction is a critical cellular process in a variety of tissues. Endothelial cells sense blood flow and transduce the mechanical stimuli into biochemical responses to adjust blood vessel diameter (Ku, 1997). Kidney epithelial cells in the collecting duct similarly sense and respond to varying rates of urine flow (Liu et al., 2003). Bone maintenance requires mechanical stimulation to maintain balanced formation and resorption (You et al., 2008). Understanding how cells sense mechanical cues and transduce them into biochemical responses is an important aspect of developing novel treatments for a wide variety of diseases of structural tissues.

Primary cilia are single immotile organelles extending from the surface of nearly all mammalian cells, and have been implicated as mechanosensors in a variety of cell types. It has been demonstrated that kidney epithelial cells respond to fluid flow, and specifically, that this mechanical stimulation causes primary cilia deflection (Praetorius and Spring, 2001). Furthermore, fluid flow initiates an intracellular calcium increase that is diminished when cilia are removed (Praetorius and Spring, 2003). Primary cilia have since been identified as mechanosensing organelles in a variety of cell types, including bone (Malone et al., 2007).

However, despite extensive efforts and the important medical implications of primary cilia function, the relation between cilium length, mechanosensitivity and gene expression has remained elusive.

SUMMARY

The presently disclosed subject matter relates to methods of regulating gene expression in a cell by modulating the length of primary cilia of the cell. In certain embodiments, the method of regulating expression of a gene in a cell comprises administering to the cell (e.g., contacting the cell with) an effective amount of one or more cilium elongation modulator, wherein the cilium elongation modulator modulates a length of one or more primary cilia of the cell.

In certain embodiments, the one or more cilium elongation modulator is contacted to the cell in an amount effective to increase the length of one or more primary cilia of the cell.

In certain embodiments, the cilium elongation modulator regulates gene expression by modulating mechanosensitivity of the cell.

In certain embodiments, the one or more cilium elongation modulator is contacted to the cell in an amount effective to increase the mechanosensitivity of the cell.

In certain embodiments, the cilium elongation modulator modulates the length of one or more primary cilia of the cell by modulating the cAMP level. In certain embodiments, the cilium elongation modulator modulates an expression level or an enzymatic activity of adenylyl cyclase, by which the cAMP level of the cell is modulated.

In certain embodiments, the cilium elongation modulator increases the length of one or more primary cilia of the cell by increasing the cAMP level.

In certain embodiments, the cilium elongation modulator increases an expression level or an enzymatic activity of adenylyl cyclase, by which the cAMP level of the cell is increased.

In certain embodiments, the cilium elongation modulator comprises one or more of fenoldopam, lithium, derivatives thereof, or combinations thereof.

In certain embodiments, the cilium elongation modulator comprises an adenylyl cyclase agonist. In certain embodiments, the cilium elongation modulator is selected from the group consisting of fenoldopam, forskolin, NKH 477 (CAS No: 138605-00-2), PACAP 1-27 (CAS No: 127317-03-7), PACAP 1-38 (137061-48-4), and combinations thereof.

In certain embodiments, the cilium elongation modulator comprises a dopamine Dl-like receptor agonist. In certain embodiments, the cilium elongation modulator is selected from the group consisting of fenoldopam, Dihydrexidine (CAS No: 158704-02- 0), Dopamine (CAS No:62-31-7), NPEC-caged-dopamine (CAS No: 1257326-23-0), SKF 38393 (CAS No:20012-10-6), SKF 77434 (CAS No:300561-58-4), SKF 81297 (CAS No:67287-39-2), SKF 82958 (CAS No:74115-01-8), SKF 83822 (CAS No:74115- 10-9), SCH-23390 (CAS No: 87075-17-0), SKF-83959 (CAS No: 67287-95-0), A68930 (CAS No: 130465-39-3), A77636 (CAS No: 145307-34-2), (R)-(-)-Apomorphine (CAS No: 314-19-2), CY 208-243 (CAS No: 100999-26-6), Ecopipam (SCH-39, 166, CAS No: 112108-01-7), and combinations thereof.

In certain embodiments, the cilium elongation modulator is lithium, derivatives thereof, or combinations thereof.

In certain embodiments, the cell being treated with the effective amount of a cilium elongation modulator is an osteocyte, an osteoblast, an osteoclast, an osteoprogenitor cell, or a combination thereof.

In certain embodiments, the gene expression modulated by contacting the cell with an effective amount of one or more cilium elongation modulator is an osteogenic gene. In certain embodiments, the one or more cilium elongation modulator is contacted to the cell in an amount effective to increase expression of one or more gene, for example, an osteogenic gene. In certain embodiments, the cilium elongation modulator is contacted to the cell in an amount effective to increase a detectable level of expression of the one or more genes by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%), 80%), 90%), 95%), 99% or more compared to a cell not contacted with the cilium elongation modulator

In certain embodiments, the osteogenic gene is selected from the group consisting of COX-2, OPN, BSP, Collagen I, Osteocalcin, and combinations thereof.

In certain embodiments, the cilium elongation modulator is contacted to a population of cells in an amount effective to increase a detectable level of expression of said one or more genes in at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of the cells.

In certain embodiments, regulating expression of the osteogenic gene causes osteogenesis.

In certain embodiments, increasing the expression of the osteogenic gene increases osteogenesis.

In certain embodiments, the cell is a mammalian cell.

In certain embodiments, the mammalian cell is a human cell.

The presently disclosed subject matter also provides for methods of treating ciliopathies. In certain embodiments, a method of treating a ciliopathy comprises administering to a subject suffering from, diagnosed as having, or at risk of having the ciliopathy, an effective amount of one or more cilium elongation modulator, which regulates expression of a gene in a cell by modulating a length of one or more primary cilia of the cell.

In certain embodiments, the one or more cilium elongation modulator is administered in an amount effective to increase the length of one or more primary cilia of the cell.

In certain embodiments, the one or more cilium elongation modulator is administered in an amount effective to increase expression of one or more gene by the cell, for example, an osteogenic gene, as descibed herein.

In certain embodiments, the cilium elongation modulator regulates gene expression by modulating mechanosensitivity of the cell. In certain embodiments, the one or more cilium elongation modulator is administered in an amount effective to increase mechanosensitivity of the cell.

In certain embodiments, increasing the one or more cilium elongation modulator is administered in an amount effective to increase expression of an osteogenic gene, and increases osteogenesis.

In certain embodiments, the ciliopathy is Alstrom syndrome, Bardet-Biedl syndrome, Joubert syndrome, Meckel-Gruber syndrome, nephronophthisis, orofaciodigital syndrome, Senior-Loken syndrome, autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD), Kartagener Syndrome, asphyxiating thoracic dysplasia, Marden-Walker syndrome, or any combination thereof.

The presently disclosed subject matter also provides for methods of treating osteoporosis. In certain embodiments, a method of treating osteoporosis comprises administering to a subject suffering from, diagnosed with, or at risk of having osteoporosis an effective amount of a cilium elongation modulator, which regulates expression of a gene in a cell by modulating a length of one or more primary cilia of the cell.

In certain embodiments, the one or more cilium elongation modulator is administered in an amount effective to increase mechanosensitivity of the cell.

The presently disclosed subject matter also provides for kits comprising one or more cilium elongation modulator, as described herein. BRIEF DESCRIPTION OF THE FIGURES

Figure 1A-1E. Small molecule treatments increase primary cilia length. 10 μΜ fenoldopam (A) and 500 μΜ lithium (B) treatment for 16 h significantly increases primary cilia length compared to vehicle control. Drug treatments elicit no change in cell viability, as assessed by MTT assay (C) and no gross morphological differences are exhibited (D,E). Mean ± SEM; n > 25 cilia for each group, n = 4 for MTT assay; ** p < 0.01, *** p < 0.001; scale bars = 20 μιη.

Figure 2A-2D. Cells with longer cilia are more mechanosensitive. Cells were subjected to fluid flow for 1 hour, and the fold change of flow vs no flow control groups was compared. Cells expressed significant increases in COX-2 (A, B) and OPN (C, D) mRNA relative to GAPDH endogenous control when treated with either fenoldopam (A, C) or LiCl (B, D) for 16 hours. Mean ± SEM; n > 5 for each group; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3. Treatment with IFT88 siRNA disrupts primary cilia formation. Overlays of primary cilia (green) and nuclei (blue) illustrate primary cilia incidence. (A) Scramble control siRNA treatment for 48 hours does not disrupt primary cilia formation. (B) IFT88 siRNA treatment results in decreased cilia length and incidence. (C) Fenoldopam treatment recovers primary cilia formation in IFT88 siRNA treated cells. Scale bars = 10 μπι.

Figure 4A-4E. Fenoldopam rescues ciliogenesis and mechanosensing. Cells treated with IFT88 siRNA have decreased cilia length compared to scramble control, while fenoldopam treatment appears to recover cilia length (n > 25 for scramble and fenoldopam treated, n = 15 for IFT88 siRNA alone) (A). Oscillatory fluid flow was applied to cells treated with IFT88 or scramble control siRNA. Impaired cilia display a decreased OPN response to fluid flow, while fenoldopam treatment is able to recover flow stimulated OPN expression (B). Treatment with IFT88 siRNA decreases cilia incidence, but is recovered with fenoldopam treatment; n > 8 fields of view (C). Fenoldopam treatment on untransfected cells has no effect on cilia incidence; n > 8 fields of view (D). Fenoldopam also did not alter IFT88 mRNA expression in untransfected cells (E). Mean ± SEM; n > 4; *** p < 0.001.

Figure 5A-5F. Fenoldopam enhances adenylyl cyclase production and activity. Cells were treated with fenoldopam or vehicle control for 16 h and then forskolin stimulated for 20 min. Fenoldopam treatment significantly increases the cAMP response to forskolin stimulation (A). Fenoldopam treatment significantly increases AC6 mRNA expression (B). AC6 knockdown decreases cilia length, but is not recovered with fenoldopam treatment (C). Neither AC6 siRNA nor fenoldopam alter cilia incidence; n > 8 fields of view (D). Oscillatory flow applied to AC6 siRNA treated cells elicits a decrease in AC6 and OPN mRNA expression and is not recovered with fenoldopam treatment (E,F). Mean ± SEM; n > 4 for each group; * p < 0.05, *** p < 0.001.

Figure 6. Osteoblast osteogenic gene expression is enhanced by culture with conditioned media from mechanically.

Figure 7A-7D. (A-C) Osteocytes with longer primary cilia are more mechanosensitive.. (D) Osteoblast osteogenic gene expression is diminished by disruption of osteocyte primary cilia formation.

Figure 8. Paracrine signaling to osteoblasts is altered by pharmacologically targeting osteocyte primary cilia-mediated mechanotransduction.

Figure 9A-9E. (A) Fenoldopam treatment enhances load-induced bone formation. (B) Load-induced bone formation assessed by dynamic histomorphometry. Alizarin (red) was administered four days after calcein (green). (C-E) Minimal adverse effects of drug treatment. There is no difference in visible bone ultrastructure between fenoldopam and vehicle control mice (Fig. 9C). Mouse weight, kidney weight, and kidney morphology assessed by H&E stain, remained unchanged in drug vs vehicle control (Fig. 9D-E). μΟΤ analysis also revealed no change in normal bone properties due to drug treatment.

Figure 10. Mice treated with cilia stiffening and lengthening agents and a TRPV4 channel agonist show signs of altered load-induced bone formation. All data are shown in reference to the non-loaded contralateral limb as control. Skeletally mature wildtype C57BL/6 mice subcutaneously injected with 4aPDD (250 μg/kg), fenoldopam (2 and 20 mg/kg), tubastatin (5 mg/kg), or vehicle control for 6 consecutive days. On day 4-6 the mice were subjected to daily axial compressive ulnar loading. Mice were placed under isoflurane anesthesia and forelimbs were placed between two loading cups controlled by an electromagnetic loading system with feedback control (Bose, ELF 3220). An initial 0.1 N preload was applied, followed by 3 N of cyclic compression applied with a 2 Hz sine wave for 120 cycles, generating a strain gage verified average peak deformation of 1895 On day 8, mice were subcutaneously injected with calcein (10 mg/kg) and alizarin red (70 mg/kg) on day 12. Mice were euthanized on day 18 and prepared for dynamic histomorphometric analysis. 4aPDD and fenoldopam treatment both displayed similar increases in bone formation rate (rBFR/BS), compared to vehicle control. Due to power limitations, tubastatin treatment did not elicit a marked change (Vehicle n = 13, tubastatin n = 3, 4aPDD n = 3, fenoldopam (2 mg/kg) n = 12, fenoldopam (20 mg/kg) n = 2, Mean ± SEM).

Figure 11. Ciliary cAMP increases following a spike in ciliary calcium influx. Osteocyte-like MLO-Y4 cells transfected with a cAMP biosensor demonstrate increased cAMP levels in the cilium in response to flow. Cells were electroporated with cAMP and calcium biosensors, seeded on collagen coated slides, treated with reduced-serum media for 3 days, and exposed to 1 Hz 10 dynes/cm² oscillatory fluid flow (OFF). A Quad-view beam splitter was used to collect ECFP, YFP, and mApple fluorescence every 0.5 seconds, simultaneously measuring ciliary cAMP and calcium influx (cAMP n=6, calcium n=8,*p < 0.05, Mean ± SEM).

Figure 12A-12B. (A) Calcium inhibition of AC6 is necessary for normal flow- induced cAMP production and osteogenic response in osteocytes. Cells transfected with mutant AC6 demonstrate increased cAMP production and decreased COX-2 response. Cells were transfected with pcDNA3.2, pcDNA3.2+AC6, or pcDNA3.2+AC6^CalMut and received 600 ug/mL Geneticin for 2 days followed by 2 days of reduced-serum media. 5 days PE, cells were placed into flow chambers, acclimated for 30 minutes, exposed to flow for 2 minutes, and lysed to quantify cAMP. RNA was isolated 1 hour after 30 minutes of OFF and COX-2 expression was quantified via RT-qPCR. Flow measurements were normalized to static controls, (n = 3, *p<0.05, Mean ± SEM). (B) Calcium binding inhibition of TRPV4 and AC6 reduce osteocyte osteogenic response to flow, while it promotes osteogenic response for AC3. Cells overexpressing TRPV4 and AC6 have increased COX-2 response to flow, but this is abrogated in mutants. AC3 overexpression has a decreased response to flow, which is also prevented by the binding pocket mutation. Data is shown as COX-2 expression of the experimental plasmid flow to no-flow response over the control plasmid flow to no-flow response. Cells were transfected with pcDNA3.2, pcDNA3.2+TRPV4, pcDNA3.2+TRPV4^CaMMut, pcDNA3.2+AC6, pcDNA3.2+AC6^CalMut, pcDNA3.2+AC3, or pcDNA3.2+AC3^CalMut and received 600 ug/mL Geneticin for 2 days followed by 2 days of reduced-serum media. 5 days PE, cells were placed into flow chambers, acclimated for 30 minutes, exposed to flow for 5 minutes. RNA was isolated 1 hour after OFF and COX-2 expression was quantified via RT-qPCR. (n = 6, *p<0.05, **p<0.01, Mean ± SEM).

Figure 13. Ciliary AC6 is depleted by disrupting a localization sequence. Cells containing a mutated intracellular VxP motif lacked AC6 localization to the cilium. Cells were transfected as in Fig 3 and immunocytochemistry (ICC) was performed (4 days PE) using primary antibodies against the V5 tag of pCDNA3.2 to visualize construct expression and Aril 3b to detect cilia (100X magnification).

Figure 14. AC3 localizes to the osteocyte cilium but is absent from kidney cilia. MLO-Y4 and IMCD cells were seeded on collagen and fibronectin coated glass bottom dishes, respectively, at similar confluences. Cells were fixed and double ICC was performed with primary antibodies against AC3 and acetylated a-tubulin to identify cilia (100X magnification).

Figure 15. Repetitive calcium peaks are seen in an osteocyte network in response to mechanical loading. Tibiae were dissected and allowed to recover in MEMa, 10% FBS, 10%) FCS for two hours and incubated with Fluo-8 AM. A preliminary sequence of 100 cyclic loads were applied (2 N preload with 8 N peak-peak amplitude). After the preload, specimens were allowed to recover for 15 mins. Then, 10 mins of 4 sec rest- inserted loading was applied and the calcium signal was measured with a 473 nm laser at the rests. (A) Osteocyte network 40 μπι below the medial proximal surface with over 40 cells imaged simultaneously (lOx objective). (B) Expanded view of inset illustrating one osteocyte with a basal calcium signal and (C) the same cell with increased signal intensity after loading, and the time-history (D) of the same osteocyte with three distinct calcium peaks.

Figure 16. Cyclic overloading of murine ulnae increases osteocyte RANKL/OPG expression indicating osteoclastogenesis. Under isofluorane anesthesia, the right ulnae were loaded, similar to Study 1.1. An initial cyclic load of 0.5 N at 2 Hz was applied to seat ulnae into the fixture. Then, 10 cycles of loading were applied at 2 N. Displacement was scaled based on previous strain gauge studies to apply approximately 3000

Displacement-controlled loading was carried out until reaching a 20% decrease in stiffness. Five days post-loading, unlae were dissected, snap frozen with liquid nitrogen, ground with a mortar and pestle and total RNA was isolated. Results are presented as the relative levels of RANKL over OPG (n=4, Mean ± SEM).

Figure 17A-17B. (A) Schematic of the TRPV4 linked calcium biosensor (TRPV4-L-CaB) construction. An Xbal restriction site is introduced to a FRET-based calcium biosensor to cut and ligate TRPV4 into the plasmid. An additional mutation after L-CaB will block an Xbal site. (B) Completed TRPV4 linked calcium biosensor. A similar strategy will be employed to add Aril 3b to TRPV4. DETAILED DESCRIPTION

The presently disclosed subject matter relates to methods of regulating gene expression in a cell by modulating the length of primary cilia of the cell. The present subject matter also provides for methods of treating ciliopathies and osteoporosis. For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections:

1. Definitions;

2. Method of Regulating Gene Expression by Modulating Cilium Length;

3. Method of Treating Ciliopathies;

4. Method of Treating Osteoporosis; and

5. Kits.

1. Definitions

The terms used in this specification generally have their ordinary meanings in the art, within the context of the present disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, e.g., up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, e.g., within 5-fold, or within 2-fold, of a value.

As used herein, the term "derivative" refers to chemical compounds with a similar or the same core structure.

As used herein, the term "cell culture" refers to a growth of cells in vitro in an artificial medium for research or medical treatment.

As used herein, the term "expressing" in relation to a gene or protein refers to generating an mRNA and/or an amino acid sequence from a nucleic acid template, for example, a gene, which can be observed using assays such as microarray assays, antibody staining assays, and the like.

As used herein, the term "ciliopathy" refers to genetic disorders caused by dysfunctional cellular cilia. Ciliopathies include, but are not limited to, Alstrom syndrome, Bardet-Biedl syndrome, Joubert syndrome, Meckel-Gruber syndrome, nephronophthisis, orofaciodigital syndrome, Senior-Loken syndrome, polycystic kidney disease (e.g. autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD), Kartagener Syndrome, asphyxiating thoracic dysplasia, Marden-Walker syndrome, and situs inversus.

As used herein, the term "osteoporosis" refers to a disease causing decreased bone strength and higher risk of a broken bone, compared to a subject that does not have osteoporosis.

As used herein, the term "treating" or "treatment" refers to clinical intervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By preventing progression of a disease or disorder, a treatment can prevent deterioration due to a disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but also a treatment may prevent the onset of the disorder or a symptom of the disorder in a subject at risk for the disorder or suspected of having the disorder.

As used herein, the terms "regulates," "modulates" or "modifies" refers to an increase or decrease in the amount, quality or effect, for example, of a particular expression of a gene, length of a cilium, or mechanosensitivity.

An "effective amount" of a substance as that term is used herein is that amount sufficient to effect beneficial or desired results, including clinical results, and, as such, an "effective amount" depends upon the context in which it is being applied. In the context of administering a composition to modulate expression of a gene, an effective amount of a composition is an amount sufficient to increase or decrease the expression of the gene. For example, the decrease can be a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%), 90%), 95%), 98%), 99% or 100% decrease in the gene expression; the increase can be a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 1000% or more increase in the gene expression. An effective amount can be administered in one or more administrations.

An "individual" or "subject" herein is a vertebrate, such as a human or non- human animal, for example, a mammal. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys.

2. Method of Regulating Gene Expression by Modulating Cilium Length

The presently disclosed subject matter relates to methods of regulating gene expression in a cell by modulating the length of primary cilia of the cell.

In certain embodiments, the methods of regulating expression of a gene in a cell comprise administering to the cell an effective amount of one or more cilium elongation modulator, wherein the cilium elongation modulator modulates a length of one or more primary cilia of the cell.

In certain embodiments, the one or more cilium elongation modulator is contacted to the cell in an amount effective to increase expression of one or more genes of the cell.

Primary cilium is a non-motile sensory cellular organelle with a 9+0 microtubule formation. Primary cilium is found on the surface of almost all mammalian cell types.

Primary cilium is assembled based on centrosome or basal bodies in quiescent cells, and it is disassembled when cells re-enter the cell cycle. Genes that control cilium assembly and cell cycle include AKT1, BBS4, CCND1, CDK5RAP2, CDKN1A (P21CIP1/WAF1), IGF1, INS2, MAP2K1, PKD1, PKD2 and TRP53. Molecule transportation within intraflagellar space and between intraflagellar space and the rest of cell plasma is important for cilium assembly and elongation. Genes essential for such transportation include, for example, DYNC2LI1, IFT172, IFT20, IFT74, IFT80, IFT88 and Kinesin-like protein (KIF3A, KTF3B). Other genes involved in cilium formation include ALMSl, ARL6, BBS1, BBS2, BBS4, BBS7, IFT172, IFT88, MKKS, OFD1, PKHDl, RPGRIPIL, VANGL2 and WWTR1. In certain embodiments, the cilium elongation modulator is an agent which modulates, for example, increases, the gene expression and/or protein level of one or more of the genes involved in cilium assembly, cell cycle, and/or intraflagellar transportation.

In certain embodiments, the cilium elongation modulator modulates the length of primary cilia of by modulating the gene expression level and/or protein level of one or more of the genes involved in cilium assembly, cell cycle, and/or intraflagellar transportation. In certain embodiments, the cilium elongation modulator increases the length of primary cilia of by increasing the gene expression level and/or protein level of one or more of the genes involved in cilium assembly, cell cycle, and/or intraflagellar transportation. In certain embodiments, the one or more gene involved in cilium assembly, cell cycle, and/or intraflagellar transportation is selected from the group consisting of AKT1, BBS4, CCND1, CDK5RAP2, CDKN1A (P21CIP1/WAF1), IGF1, INS2, MAP2K1, PKD1, PKD2, TRP53. DYNC2LI1, IFT172, IFT20, IFT74, IFT80, TRPV4, IFT88, Kinesin-like protein (KIF3A, KIF3B), ALMSl, ARL6, BBS1, BBS2, BBS4, BBS7, IFT172, IFT88, MKKS, OFDl, PKHDl, RPGRIPIL, VANGL2 and WWTR1.

Primary cilia maintain lengths from 1 to 10 μιη in mammalian cells. Intracellular signaling pathways can affect cilia length control. One cilium length control effector is cAMP and its associated calcium signaling pathway. Increased cyclic AMP levels and PKA activity in cells can stimulate the growth of cilia. Adenylate cyclase (AC) knockdown or reduction can block or inhibit cilium length increase.

Lithium, an inhibitor of glycogen synthase kinase 3β (GSK3P) can cause cilium elongation by decreasing GSK3P activity. The NIMA related kinase (Nek) family kinases are also involved in regulating cilia length control and congenital mutations on them cause ciliopathies. Nek family kinases have 11 members and Nekl and 8 can also influence cilium growth.

Signal transduction pathways are also involved in cilia length control at different steps of ciliogenesis, including, but not limited to, FGF signaling. The Mammalian target-of-rapamycin (mTOR) pathways can also modulate ciliary length. Suppression of mTOR pathway by rapamycin can result in short cilia and pathway activation increases the length of cilia. In addition, MAK, CCRK CDC14 also affects ciliogenesis.

Cell shape and contractility genes can also determine cilium length. For example, polymerization of actin filaments suppresses cilia formation. Gelsolin family members, e.g., GSN and AVIL, can promote cilia formation. ACTR3, which polymerizes actin filaments, can inhibit ciliation, while its depletion can increase cilium length. Likewise, cytochalasin D, a drug that inhibits actin filament polymerization, can increase the length of the cilia. Jasplakinolide, a drug that induces the formation of actin filaments, can also lead to an increase in cilium length. Additionally the orientation of the actin cytoskeleton and the level of stress fiber formation can have a significant impact on cilium length.

In certain embodiments, the one or more cilium elongation modulator is administrated in a effective amount to modulate the gene expression of one or more signal pathway genes associated with primary cilia. Primary cilia are a nexus of cell signaling, associated with regulation of the Notch, Hedgehog, PDGF, TOR and Wnt signaling pathways. Components of these signaling pathways are concentrated within the ciliary compartment, which promotes efficient signal transduction. Signal pathway genes associated with primary cilia include

• Hedgehog pathway: BTRC (BTRCP), FUZ, GLI1, GLI2, GLI3, GSK3B, IHH, INTU, LRP2, PTCH1, RAB23, SHH, SMO and SUFU;

• cAMP pathway : ADCY3, ADCY7, AVPR2, HTR6, PKD2 and SSTR3;

• mTOR pathway: AKT1, CDC42, GSK3B, IGF1, INS2, MAPKl, MTOR, PIK3CA (P110A), PRKCA, RHOA, TRP53, TSC1 and TSC2;

• Planar Cell Polarity pathway: DVLl, FAT4, FJX1, FUZ, FZD1, INTU, RHOA, ROCK2, VANGL2 and WNT9B;

• WNT pathway: AXIN2, GSK3p and INVS; and

• MAP Kinase pathways: FOS, KRAS, MAP2K1 (MEK1), MAPKl (ERK2), MOS, PRKCA and PTPN5.

In certain embodiments, the one or more cilium elongation modulator regulates gene expression by modulating mechanosensitivity of the cell. In certain embodiments, the deflection of the primary cilium can cause an increase in intracellular calcium. Such calcium response can be mediated by a mechanosensory complex located at the base of the cilium, comprising Polycystin 1 and Polycystin 2. For example, the primary cilium can function as a mechanosensor in bone cells, for example, osteocytes, osteoblasts, osteoclasts, osteoprogenitor cells, or a combinations thereof, where deflection of the primary cilium, for example, under fluid flow can increase expression of osteogenic genes as described herein, for example, COX-2 and/or OPN. In certain embodiments, deflecting a primary cilium induces a rapid and transient decrease in cAMP. In certain embodiments, the cilium elongation modulator modulates the length of one or more primary cilia of the cell by modulating, for example, decreasing, cAMP level or activity.

Cyclic adenosine monophosphate (cAMP or cyclic AMP) is a derivative of adenosine triphosphate (ATP) and is involved in intracellular signal transduction. Cyclic AMP is synthesized by adenylate cyclase, a 12-transmembrane glycoprotein that catalyzes ATP to form cAMP. The cAMP produced is a second messenger in cellular metabolism and is an allosteric activator of protein kinase A (PKA). Adenylate cyclase is activated by signaling molecules through the activation of adenylate cyclase stimulatory G-protein-coupled receptors. Adenylate cyclase is inhibited by agonists of adenylate cyclase inhibitory G protein-coupled receptors. cAMP signals can be terminated by cAMP phosphodiesterase, an enzyme that degrades cAMP and inactivates protein kinase A.

In certain embodiments, the cilium elongation modulator modulates, for example, decreases, an expression level or an enzymatic activity of adenylyl cyclase, by which the cAMP level or activity in the cell is modulated.

In certain embodiments, the cilium elongation modulator comprises fenoldopam, lithium, derivatives thereof, or combinations thereof.

In certain embodiments, the cilium elongation modulator comprises an adenylyl cyclase agonist. In certain embodiments, the cilium elongation modulator is selected from the group consisting of fenoldopam, forskolin, NKH 477 (CAS No: 138605-00-2), PACAP 1-27 (CAS No: 127317-03-7), PACAP 1-38 (137061-48-4), and combinations therod.

In certain embodiments, the cilium elongation modulator comprises a dopamine Dl-like receptor agonist. In certain embodiments, the cilium elongation modulator is selected from the group consisting of fenoldopam, Dihydrexidine (CAS No: 158704-02- 0), Dopamine (CAS No:62-31-7), NPEC-caged-dopamine (CAS No: 1257326-23-0), SKF 38393 (CAS No:20012-10-6), SKF 77434 (CAS No:300561-58-4), SKF 81297 (CAS No:67287-39-2), SKF 82958 (CAS No:74115-01-8), SKF 83822 (CAS No:74115- 10-9), SCH-23390 (CAS No: 87075-17-0), SKF-83959 (CAS No: 67287-95-0), A68930 (CAS No: 130465-39-3), A77636 (CAS No: 145307-34-2), (R)-(-)-Apomorphine (CAS No: 314-19-2), CY 208-243 (CAS No: 100999-26-6), Ecopipam (SCH-39, 166, CAS No: 112108-01-7), and combinatiosn thereof.

In certain embodiments, the cilium elongation modulator is lithium. In certain embodiments, the cilium elongation modulator modulates the length of primary cilia of by modulating the gene expression level and/or protein level of one or more of the genes involved in cilium assembly, cell cycle, and/or intraflagellar transportation. In certain embodiments, the cilium elongation modulator increases the length of primary cilia by increasing the gene expression level and/or protein level of one or more of the genes involved in cilium assembly, cell cycle, and/or intraflagellar transportation. In certain embodiments, the one or more gene involved in cilium assembly, cell cycle, and/or intraflagellar transportation is selected from the group consisting of AKT1, BBS4, CC D1, CDK5RAP2, CDKN1A (P21CIP1/WAF1), IGF1, INS2, MAP2K1, PKD1, PKD2, TRP53. DYNC2LI1, IFT172, IFT20, IFT74, IFT80, TRPV4, IFT88, Kinesin-like protein (KIF3A, KIF3B), ALMSl, ARL6, BBS1, BBS2, BBS4, BBS7, IFT172, IFT88, MKKS, OFDl, PKHDl, RPGRIPIL, VANGL2 and WWTR1.

In certain embodiments, the cilium elongation modulator comprises a nucleic acid sequence encoding one or more proteins, or functional fragments thereof, involved in cilium assembly, cell cycle, and/or intraflagellar transportation, signal transduction, cell shape and contractility, as described herein.

In certain embodiments, the cilium elongation modulator comprises an amino acid sequence of a protein, or functional fragment thereof, involved in cilium assembly, cell cycle, and/or intraflagellar transportation, signal transduction, cell shape and contractility, as described herein.

In certain embodiments, the cilium elongation modulator decreases the length of primary cilia of by decreasing the gene expression level and/or protein level of one or more of the genes involved in cilium assembly, cell cycle, and/or intraflagellar transportation. In certain embodiments, the cilium elongation modulator comprises a compound that can decrease expression or activity of one or more genes or proteins involved in cilium assembly, cell cycle, and/or intraflagellar transportation, signal transduction, cell shape and contractility, as described herein. Such compounds can include, for example, an RNAi molecule, antibody or fragment thereof capable of targeting one or more gene involved in cilium assembly, cell cycle, and/or intraflagellar transportation, signal transduction, cell shape and contractility. Examples of RNAi molecules include, but are not limited to, the following: siRNA, shRNA, microRNA, double stranded RNA, as well as any modifications or derivatives thereof. Modulation of gene expression can be accomplished by a recombinant DNA construct. In certain embodiments, a vector (e.g., a non-viral vector or a rival vector, e.g., a gamma-retroviral or lentiviral vector) can be employed for the introduction of the DNA construct into the cell. For example, a polynucleotide encoding a genes involved in cilium assembly, cell cycle, and/or intraflagellar transportation can be cloned into a vector and expression can be driven from a endogenous promoter of the vector, or from a promoter specific for a target cell type of interest.

In certain embodiments, other viral vectors are used to modulate the expression of genes involved in cilium assembly, cell cycle, and/or intraflagellar transportation, for example, adenoviral, lentiviral, and adeno-associated viral vectors, vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244: 1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1 :55-61, 1990; Sharp, The Lancet 337: 1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995).

In certain embodiments, non-viral approaches are used to modulate the expression of genes involved in cilium assembly, cell cycle, and/or intraflagellar transportation. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101 :512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263 : 14621, 1988; Wu et al., Journal of Biological Chemistry 264: 16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science 247: 1465, 1990).

In certain embodiments, other non-viral means are used to modulate the expression of genes involved in cilium assembly, cell cycle, and/or intraflagellar transportation. Other non-viral means for gene transfer include transfection in vitro using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a subject can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue or are injected systemically. Transient expression may be obtained by RNA electroporation. cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element or intron (e.g. the elongation factor lc enhancer/promoter/intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

In addition to full-length polypeptides, the present disclosure also provides fragments of any one of the polypeptides or peptide domains of the genes involved in cilium assembly, cell cycle, and/or intraflagellar transportation. As used herein, the term "a fragment" means at least 5, 10, 13, or 15 amino acids. In other embodiments a fragment is at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids, and in other embodiments at least 60 to 80, 100, 200, 300 or more contiguous amino acids. Fragments of the genes can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

In certain embodiments, the cell being contacted with a cilium elongation modulator is an osteocyte, an osteoblast, an osteoclast, an osteoprogenitor cell, or combinations thereof.

In certain embodiments, the cilium elongation modulator is contacted to a population of cells.

Osteocytes are bone cells located in mature bone. Osteocytes are derived from osteoprogenitor cells. Osteocytes generate bone matrix through mechanosensory mechanisms. Osteocytes decompose bone through a rapid, transient mechanism, i.e., osteocytic osteolysis, and deposit hydroxyapatite, calcium carbonate and calcium phosphate. Osteocytes also synthesize sclerostin, a secreted protein product of the SOST gene that inhibits bone formation by binding to LRP5/LRP6 receptors and blocking Wnt signaling. Sclerostin is inhibited by parathyroid hormone (PTH) and mechanical loading. Sclerostin inhibits the activity of BMP (bone morphogenetic protein).

Osteoblasts are another type of bone cells. Osteoblasts synthesize dense, crosslinked collagen, and also synthesize osteocalcin and osteopontin, which comprise the matrix of bone.

Osteoclasts are a type of bone cells responsible for the breakdown and remodeling of bones. An osteoprogenitor cell is the precursor of the differentiated bone cells described above. Unlike the other types of bone cells, osteoprogenitor cells maintain the ability to divide.

In certain embodiments, the expression of the one or more gene modified according to the methods described herein is an osteogenic gene. Osteogenic genes include, but are not limited to, genes that promote osteogenesis. Non-limiting examples of osteogenic genes include the following:

• Runt-related transcription factor 2 (Runx2, (Cbfal/PEBP2aA/AML-3/Osf2)), Osterix (Osx), distal-less homeobox protein 5 (Dlx5), Alkaline phosphatase (ALP), Msx-2 (Hox-8), Nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κΒ), Osteoprotegerin (OPG), Cytochrome c oxidase subunit 2 (Cox-2), fibroblast growth factor 2 (FGF2), bagpipe homeobox homolog 1 (Drosophila) (Bapxl), Collagen I, Osteocalcin, Osteopontin (OPN), and Bone sialoprotein (BSP).

• Bone mineralization genes: AHSG, AMBN, AMELY, BGLAP, ENAM, MINPPl, STATH, and TUFT1.

• Cartilage condensation genes: BMP1, COL11 Al, and SOX9.

• Ossification genes: ALPL, AMBN, AMELY, BGLAP, CALCR, CDH11, DMP1, DSPP, ENAM, MINPPl, PHEX, RUNX2, STATH, TFIP11, and TUFT1.

• Calcium ion binding and homeostasis genes: ANXA5, BGLAP, BMP1, CALCR, CDH11, COMP, DMP1, EGF, MMP2, MMP8, and VDR.

• Growth factors and receptors: BMP1, BMP2, BMP3, BMP4, BMP 5, BMP6, CSF2, CSF3, EGF, EGFR, FGF1, FGF2, FGF3, FGFR1, FGFR2, FLT1, GDF10, IGFl, IGFIR, IGF2, PDGFA, TGFB 1, TGFB2, TGFB3, TGFBR1, TGFBR2, VEGFA, and VEGFB.

• Extracellular matrix (ECM) Molecules: COL4A3, COL10A1, COL11A1, COL12A1, COL14A1, COL15A1, COL1A1, COL1A2, COL2A1, COL3A1,

COL4A3, COL5A1.

• ECM protease inhibitors: AHSG, COL4A3, and SERPINH1

• ECM proteases: CTSK, MMP10, MMP2, MMP8, MMP9, and PHEX.

• Cell Adhesion Molecules: CDH11, COL11A1, COL14A1, ICAM1, ITGB 1, VCAM1, ITGA1, ITGA2, ITGA3, ITGAM, ITGB1, BGLAP, CD36, COL12A1,

COL15A1, COL4A3, COL5A1, COMP, FN1, SCARBl,and TNF.

• Transcription factors: MSX1, FKB1, RUNX2, SMAD1, SMAD2, SMAD3, SMAD4, SOX9, TNF, TWIST 1, and VDR.

In certain embodiments, the cilium elongation modulator increases the length of one or more primary cilia of the cell. In certain embodiments, the cilium elongation modulator increases an expression level of one or more osteogenic gene by increasing the length of one or more primary cilia of the cell. In certain embodiments, the one or more osteogenic gene is selected from the group consisting of COX-2, OPN, BSP, Collagen I, Osteocalcin, Runt-related transcription factor 2 (Runx2, (Cbfal/PEBP2aA/AML-3/Osf2)), Osterix (Osx), distal-less homeobox protein 5 (Dlx5), Alkaline phosphatase (ALP), Msx-2 (Hox-8), Nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κΒ), Osteoprotegerin (OPG), Cytochrome c oxidase subunit 2 (Cox-2), fibroblast growth factor 2 (FGF2), bagpipe homeobox homolog 1 (Drosophila) (Bapxl), Collagen I, Osteocalcin, Osteopontin (OPN), Bone sialoprotein (BSP), AHSG, AMBN, AMELY, BGLAP, ENAM, MINPPl, STATH, TUFT1, Cartilage condensation genes: BMP1, COL11A1, SOX9, ALPL, AMBN, AMELY, BGLAP, CALCR, CDH11, DMP1, DSPP, ENAM, MINPPl, PHEX, RUNX2, STATH, TFIP11, TUFT1, ANXA5, BGLAP, BMP1, CALCR, CDH11, COMP, DMP1, EGF, MMP2, MMP8, VDR, BMP1, BMP2, BMP3, BMP4, BMP 5, BMP6, CSF2, CSF3, EGF, EGFR, FGFl, FGF2, FGF3, FGFRl, FGFR2, FLTl, GDF10, IGFl, IGFIR, IGF2, PDGFA, TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, VEGFA, VEGFB, COL4A3, COL10A1, COL11A1, COL12A1, COL14A1, COL15A1, COL1A1, COL1A2, COL2A1, COL3A1, COL4A3, COL5A1, AHSG, COL4A3, SERPINHl, CTSK, MMP10, MMP2, MMP8, MMP9, and PHEX, CDH11, COL11A1, COL14A1, ICAM1, ITGB 1, VCAM1, ITGA1, ITGA2, ITGA3, ITGAM, ITGB 1, BGLAP, CD36, COL12A1, COL15A1, COL4A3, COL5A1, COMP, FN1, SCARB1, TNF, MSX1, NFKB 1, RUNX2, SMADl, SMAD2, SMAD3, SMAD4, SOX9, TNF, TWIST 1, VDR, and combinations thereof.

In certain embodiments, the osteogenic gene is selected from the group consisting of COX-2, OPN, BSP, Collagen I, Osteocalcin, and combinations thereof. In certain embodiments, increasing expression of one or more osteogenic genes increases osteogenesis. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the mammalian cell is a human cell.

3. Method of Treating Ciliopathies

The presently disclosed subject matter is also directed to methods of treating ciliopathies. Human congenital disruption of cilium structure or function can cause developmental disorders. Many diseases are attributed to cilia formation and/or functional defects, which affect organs, such as kidney, brain, limb, eye, ear, liver and bone. Identified ciliopathies include, but are not limited to, Joubert syndrome (JBTS), nephronophthisis (NPHP), autosomal dominant and recessive polycystic kidney disease (ADPKD and ARPKD), Meckel-Gruber syndrome (MKS), Bardet-Biedl syndrome (BBS), Alstrom syndrome, orofaciodigital syndrome, Senior-Loken syndrome, Kartagener Syndrome, asphyxiating thoracic dysplasia, and Marden-Walker syndrome among others.

In certain embodiments, a method of treating a ciliopathy comprises administering to a subject suffering from, diagnosed with, or at risk of having a ciliopathy, an effective amount of a cilium elongation modulator, as described herein, that regulates expression of a gene in a cell of the subject by modulating a length of one or more primary cilia of the cell.

In certain embodiments, the cilium elongation modulator is administered in an amount effective to increase length of one or more primary cilia of the cell.

In certain embodiments, the cilium elongation modulator is administered in an amount effective to increase expression of a gene in the cell, wherein the gene, for example, comprises one or more osteogenic genes.

In certain embodiments, the cilium elongation modulator is administered in an amount effective to increase ostrogenesis. In certain embodiments, the ciliopathy is Alstrom syndrome, Bardet-Biedl syndrome, Joubert syndrome, Meckel-Gruber syndrome, nephronophthisis, orofaciodigital syndrome, Senior-Loken syndrome, autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD)), Kartagener Syndrome, asphyxiating thoracic dysplasia, Marden-Walker syndrome, or any combination thereof.

In certain embodiments, the cilium elongation modulator comprises one or more agents which modulate gene expression and/or protein level of one or more of the genes involved in cilium assembly, cell cycle, and/or intraflagellar transportation, as described herein. In certain embodiments, the cilium elongation modulator increases the length of primary cilia of by increasing the gene expression level and/or protein level of one or more of the genes involved in cilium assembly, cell cycle, and/or intraflagellar transportation. In certain embodiments, the one or more gene involved in cilium assembly, cell cycle, and/or intraflagellar transportation is selected from the group consisting of AKT1, BBS4, CC D1, CDK5RAP2, CDKN1A (P21CIP1/WAF1), IGF1, INS2, MAP2K1, PKD1, PKD2, TRP53. DYNC2LI1, IFT172, IFT20, IFT74, IFT80, TRPV4, IFT88, Kinesin-like protein (KIF3A, KIF3B), ALMSl, ARL6, BBS1, BBS2, BBS4, BBS7, IFT172, IFT88, MKKS, OFDl, PKHDl, RPGRIPIL, VANGL2 and WWTR1.

The presently disclosed cilium elongation modulator can be administered in any physiologically acceptable vehicle. Pharmaceutical compositions comprising the presently disclosed cilium elongation modulator and a pharmaceutically acceptable carrier are also provided. The presently disclosed cilium elongation modulator and the pharmaceutical compositions comprising thereof can be administered via localized injection, orthotopic (OT) injection, systemic injection, intravenous injection, or parenteral administration. In certain embodiments, the presently disclosed cilium elongation modulator are administered to a subject suffering from a ciliophacy via systemic or localized injection.

The presently disclosed cilium elongation modulator and the pharmaceutical compositions comprising thereof can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof. Sterile injectable solutions can be prepared by incorporating the compositions of the presently disclosed subject matter, e.g., a composition comprising the presently disclosed cilium elongation modulator, in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, alum inurn monostearate and gelatin. According to the presently disclosed subject matter, however, any vehicle, diluent, or additive used would have to be compatible with the presently disclosed cilium elongation modulators.

Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose can be used because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The concentration of the thickener can depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form).

Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the presently disclosed cilium elongation modulator. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.

An "effective amount" (or "therapeutically effective amount") is an amount sufficient to affect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the ciliopathy, or otherwise reduce the pathological consequences of the ciliopathy. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form and effective concentration of the cells administered.

In certain embodiments, an effective amount of the presently cilium elongation modulator is an amount that is sufficient to reduce or eliminate the symptoms of a subject suffering from a ciliopathy. In certain embodiments, an effective amount of the presently disclosed cilium elongation modulator is an amount that is sufficient to prevent a subject from developing a ciliopathy.

4. Method of Treating Osteoporosis

The presently disclosed subject matter also provides for methods of treating osteoporosis. In certain embodiments, a method of treating osteoporosis comprises administering to a subject suffering from, diagnosed with, or at risk of having osteoporosis, an effective amount of a cilium elongation modulator, as described herein, that regulates expression of a gene in a cell by modulating a length of one or more primary cilia of the cell. In certain embodiments, the cilium elongation modulator is administered in an amount effective to increase length of one or more primary cilia of the cell.

In certain embodiments, the cilium elongation modulator is administered in an amount effective to increase ostrogenesis.

Osteoporosis is a disease causing decreased bone strength and higher risk of a broken bone. Commonly affected bones include the back bones, the bones of the forearm, and the hip. There are typically no symptoms until a broken bone occurs. The WNT/Lrp pathway is a regulator of bone anabolism. In certain embodiments, the cilium elongation modulator is administered in an amount effective to increase bone strength and/or reduce risk of a broken bone, compared to a subject with osteoporosis that is not administered the cilium elongation modulator.

In certain embodiments, the cilium elongation modulator comprises one or more agents that modulates the gene expression and/or protein level of one or more of the genes involved in cilium assembly, cell cycle, and/or intraflagellar transportation, as described herein. In certain embodiments, the cilium elongation modulator increases the length of primary cilia of by increasing the gene expression level and/or protein level of one or more of the genes involved in cilium assembly, cell cycle, and/or intraflagellar transportation. In certain embodiments, the one or more gene involved in cilium assembly, cell cycle, and/or intraflagellar transportation is selected from the group consisting of AKT1, BBS4, CCND1, CDK5RAP2, CDKN1A (P21CIP1/WAF1), IGF1, INS2, MAP2K1, PKD1, PKD2, TRP53. DYNC2LI1, IFT172, IFT20, IFT74, IFT80, TRPV4, IFT88, Kinesin-like protein (KIF3A, KIF3B), ALMSl, ARL6, BBS1, BBS2, BBS4, BBS7, IFT172, IFT88, MKKS, OFDl, PKHDl, RPGRIPIL, VANGL2 and WWTR1.

The presently disclosed cilium elongation modulator and the pharmaceutical compositions comprising thereof can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof. Sterile injectable solutions can be prepared by incorporating the compositions of the presently disclosed subject matter, e.g., a composition comprising the presently disclosed stem-cell-derived precursors, in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, alum inurn monostearate and gelatin. According to the presently disclosed subject matter, however, any vehicle, diluent, or additive used would have to be compatible with the presently disclosed cilium elongation modulator.

An "effective amount" (or "therapeutically effective amount") is an amount sufficient to affect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of osteoporosis, or otherwise reduce the pathological consequences of osteoporosis. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form and effective concentration of the cells administered.

In certain embodiments, an effective amount of the presently cilium elongation modulator is an amount that is sufficient to reduce or eliminate the symptoms of a subject suffering from Osteoporosis. In certain embodiments, an effective amount of the presently disclosed cilium elongation modulator is an amount that is sufficient to prevent a subject from developing Osteoporosis.

5. Kits

The presently disclosed subject matter also provides for kits for regulating gene expression in a cell by modulating the length of the primary cilia of the cell. In certain embodiments, kits are provided for increasing the length of the cell's primary cilia, and increasing expression of one or more genes of the cell, for example, one or more osteogenic genes, as described herein. In certain embodiments, kits are provided for increasing the production of bone by a cell, for example, an osteoblast. In certain embodiments, the kits comprise one or more cilium elongation modulator, as described herein. In certain embodiments, the kits also comprise instructions for modulating the length of primary cilia of a cell, and thereby regulating expression of a gene in a cell, for example, increasing the length of the cell's primary cilia and increasing the expression of one or more osteogenic gene.

EXAMPLES

The presently disclosed subject matter will be better understood by reference to the following Example, which is provided as exemplary of the presently disclosed subj ect matter, and not by way of limitation.

Example 1: Lengthening primary cilia enhances cellular mechanosensitivity

Osteocytes are mechanosensitive cells within bone, where the primary cilium functions as a mechanosensor in this context (Malone et al., 2007). In vitro, fluid flow mechanical stimulation of osteocytes enhances expression of the osteogenic genes cyclooxygenase-2, COX-2, and osteopontin, OPN. COX-2 synthesizes prostaglandin E2, and OPN is a critical extracellular matrix protein. Increases in the production of both are indicative of osteogenesis (Ehrlich and Lanyon, 2002; Fujihara et al., 2006; Klein- Nulend et al., 1997; Raisz, 1999). When osteocyte primary cilia formation is inhibited, the cells display an abrogated osteogenic response to flow, implicating the cilium as a critical mechanosensor in osteocytes (Malone et al., 2007). Furthermore, it has been previously reported that adenylyl cyclases, specifically AC6, play a significant role in osteocyte mechanosensitivity (Kwon et al., 2010). Adenylyl cyclases convert ATP to the ubiquitous second messenger cAMP, a process which can be specifically stimulated by forskolin.

Due to the significance of primary cilia in cellular mechanotransduction, the inventors hypothesized that increasing their length would enhance mechanosensitivity. Here, the inventors treated osteocytes with lithium and fenoldopam to increase ciliary length, and then mechanically stimulate the cells (Kathem et al., 2014; Miyoshi et al., 2009; Upadhyay et al., 2014). The results highlight the importance of cilium length in cellular mechanosensitivity, and that this is a process that can be modulated by pharmacologic intervention.

(1) Modulation of Primary Cilia Length

To test the hypothesis that cilium length directly affects mechanosensitivity, the inventors first verified that primary cilia length could be modulated. MLO-Y4 osteocytes were cultured and treated with two distinct small molecules to increase cilia length. Cells were cultured in media supplemented with fenoldopam, lithium, or vehicle control for 16 hours, and no gross morphological changes resulted from the drug treatments. Immunocytochemistry was then used to image primary cilia. Both fenoldopam and lithium treatments induced significant increases in cilia length by 26% ± 7% and 46% ± 5%), respectively, compared to vehicle control (Fig. 1A, B). Cell viability was assessed with MTT assay and it found no change upon drug treatments (Fig. 1C). Additionally, no gross morphological changes resulted from fenoldopam or lithium treatment (Fig. ID, E).

(2) Effect of Elongating Cilia on Cellular Mechanosensitivity Next, the inventors examined the effect of elongating cilia on cellular mechanosensitivity by mechanically stimulating cells with longer cilia and analyzing their osteogenic response. Osteocytes were treated with fenoldopam, lithium, or vehicle control, and exposed to oscillatory fluid flow for 1 hr. As a control, samples were simultaneously loaded into flow chambers, but not subjected to flow. Mechanosensitivity was then quantified at the mRNA level with analysis of COX-2 and OPN expression, and presented as the fold change of flow over no flow control (Fig. 2). Cells with cilia lengthened by fenoldopam were more responsive, exhibiting elevated mRNA expression of 124% ± 27% and 48% ± 8% of COX-2 and OPN respectively compared to unlengthened controls. Lithium resulted in a more modest, but still significant increase in response of 61% ± 13% and 34% ± 8% for COX-2 and OPN. This flow-induced enhanced osteogenic response was observed in cells with elongated primary cilia, regardless of the means of lengthening, suggesting that the effect was due to lengthening and not an unanticipated effect of the agents utilized.

(3) Effect of Primary Cilia Length to Cilia Function.

The inventors next sought to examine the potential of targeting primary cilia length to recover impaired cilia function. IFT88 inhibition was employed as a model of dysfunctional cilia, and has previously been used to mimic the effects of polycystic kidney disease (Lehman et al., 2008). IFT88 is a critical component of intraflagellar transport and is necessary for proper primary cilia formation (Pazour et al., 2000; Yoder et al., 2002). Cells treated with IFT88 siRNA displayed decreased primary cilia length and incidence compared to scramble control (Fig. 3A, B). IFT88 siRNA treated cells were then treated with fenoldopam, and cilia length and incidence were noticeably recovered (Fig. 3C). Upon analysis, cilia of IFT88 siRNA treated cells were significantly shorter (Fig. 4A) and were present with lower incidence (Fig. 4C) than scramble control groups, with fenoldopam treatment significantly recovering cilia incidence. Then the cells were mechanically stimulated to examine whether ciliogenesis recovery restored mechanosensitivity. Fluid flow was applied for 1 h and cells with impaired primary cilia formation displayed significantly decreased flow-induced OPN mRNA expression by 43 % ± 2 %, compared to scramble control (Fig. 4B). Then, fenoldopam treatment was able to recover this OPN response by 52 % ± 1 %, compared to IFT88 siRNA and vehicle treated cells. This further suggests that the length of primary cilia is critical to their function as a mechanosensor and that as cilia formation was restored, so was mechanosensitivity too. Then it was confirmed that fenoldopam treatment of healthy, ciliated cells had no significant effect on cilia incidence (Fig. 4D) or IFT88 mRNA expression (Fig. 4E).

(4) Molecular Pathway Through Which Fenoldopam Increases Primary Cilia

Length

Finally, the inventors examined a potential molecular pathway through which fenoldopam increases primary cilia length. cAMP has been previously shown to be involved in ciliogenesis and primary cilia-mediated mechanotransduction, so adenylyl cyclase activity was quantified by measuring stimulated cAMP production (Besschetnova et al., 2010; Kwon et al., 2010). The inventors increased primary cilia length by fenoldopam treatment, and then briefly stimulated the cells with the adenylyl cyclase agonist, forskolin (Fig. 5A). Fenoldopam treatment significantly enhanced the forskolin stimulated cAMP response by 130% ± 25% compared to vehicle control.

Additionally, fenoldopam treatment stimulated a 20% ± 8% increase in AC6 mRNA expression (Fig. 5B). Because of the significant role of AC6 in primary cilia-mediated mechanotransduction, inhibition of AC6 was used as an alternative model of impaired cell mechanosensitivity. Treatment of osteocytes with AC6 siRNA resulted in a small but significant decrease in cilia length by 10 % ± 3 % (Fig. 5C), while AC6 inhibition had no effect on cilia incidence (Fig. 5D). Fenoldopam had no effect on recovering cilia length or incidence in AC6 siRNA treated cells. When cells with diminished AC6 were mechanically stimulated, AC6 knockdown cells displayed decreased AC6 and flow- induced OPN expression by 50 % ± 3 % and 30 % ± 3 %, respectively, which was not recovered with fenoldopam treatment (Fig. 5E, F).

(5) Materials and Methods

(5-1) Cell culture and drug treatments

MLO-Y4 osteocytes were cultured on collagen-coated dishes in MEMa (Life Tech) supplemented with 5% fetal bovine serum, 5% calf serum, and 1% penicillin/streptomycin at 37°C and 5% C02. Fenoldopam mesylate (Sigma) was used at 10 μΜ diluted in DMSO, dimethyl sulfoxide, (Sigma) and normal culture media. Lithium chloride (Sigma) was used at 500 μΜ diluted in normal culture media - a dose response from 50 μΜ to 10 mM was examined with 500 μΜ being the lowest dose to increase length significantly (data not shown). These agents, or their vehicle control, were applied to cells for 16 h prior to experimentation. MTT, (methylthiazolyldiphenyl- tetrazolium bromide) assay (Sigma) was performed according to manufacturer's protocol to assess cell viability during drug treatments. Phase contrast microscopy with an Olympus CKX41 inverted microscope and 40 ^χ objective was used to assess cell morphology.

(5-2) Immunocytochemistry

For primary cilia imaging and analysis, cells cultured on collagen I-coated glass were fixed in 10 % formalin and treated with anti-acetylated a-tubulin primary antibody, 1 : 1, from a C3B9 hybridoma cell line (Sigma). Cilia were visualised with Alexa-Fluor 488 secondary antibody, 1 : 1000 (Life Technologies) and imaged with a Ι ΟΟ^χ oil objective on an Olympus Fluoview F VI 000 confocal microscope. Nuclei were stained with DAPI (Life Technologies). Cilia length was analyzed using Image J software. (5-3) Oscillatory fluid flow

Cells were exposed to oscillatory fluid flow as a mechanical stimulus. Cells were seeded on collagen I-coated glass slides at ~ 2,800 cells/cm² and cultured for 72 h before application of flow. Drug treatments were applied 16 h prior to experimentation. Slides were loaded into parallel plate flow chambers (dimensions: 75 ^χ 38 ^χ 0.28 mm) and allowed to incubate at 37 °C for 30 min prior to initiation of stimulation (Kwon et al, 2010; Lee et al, 2014; Mai one et al , 2007). Flow was applied for 1 h at 1 Hz with a peak flow rate of 18.8 mL/min, providing 1 Pa peak wall shear stress.

(5-4) mRNA expression

Immediately after flow, cells were washed with PBS and total mRNA was isolated using TriReagent (Sigma). Total mRNA was converted to cDNA by TaqMan reverse transcriptase (Applied Biosystems). Gene expression was analyzed by quantitative real-time PCR using primers and probes (Life Technologies) for analysis of cyclooxygenase-2, COX-2 (Mm00478374_ml); osteopontin, OPN (Mm00436767_ml); adenylyl cyclase 6, AC6 (Mm00475772_ml); intraflagellar transport 88, IFT88 (Mm00493675_ml); and GAPDH (4351309). Samples and standards were run in triplicate, and all gene expression was normalized to GAPDH endogenous control.

(5-5) RNA interference

Gene silencing was performed by siRNA mediated knockdown and compared to scramble siRNA control (Life Technologies). For primary cilia disruption, cells were transfected with 20 μΜ IFT88 siRNA (5 '-CCAGAAAC AGATGAGGACGACCTTT-3 ') (SEQ ID NO: l), AC6 siRNA (5'-CCTGCCACCTACAACAGCTCAATTA-3 ') (SEQ ID NO:2), or scrambled siRNA control using Lipofectamine 2000 (Life Technologies) as previously described (Kwon et al , 2010).

(5-6) Adenylyl cyclase activity

Adenylyl cyclase activity was quantified by cAMP ELISA (Enzo). Cells were cultured as previously described and treated with 10 μΜ fenoldopam for 16 hours. Cells were stimulated by 10 μΜ forskolin (Sigma) or DMSO vehicle control for 20 minutes prior to lysis with 0.1 M HC1. Cell lysate was analyzed according to manufacturer's protocol, and normalized to total protein quantified by BCA (Thermo Fisher). All samples and standards were run in duplicate.

(5-7) Statistic Analysis

All data were analyzed with one-way ANOVA followed by Bonferroni post-hoc correction. Values are reported as mean ± SEM, with p < 0.05 considered statistically significant. Sample size, n, represents biological replicates.

(6) Discussion

The results demonstrated that primary cilia length plays a significant role in cell mechanosensitivity. Two distinct, clinically utilized, small molecules were employed to increase cilia length and both resulted in enhanced mechanosensitivity. Cells with impaired ciliogenesis had impaired mechanosensing, but this could be recovered with fenoldopam treatment. Finally, The results showed that fenoldopam modulates osteocyte mechanosensitivity through a mechanism involving AC6 and cells with diminished AC6 had shorter cilia and impaired mechanosensing.

Based on clinical and biochemical considerations, fenoldopam was a more suitable candidate for further study. Fenoldopam is a dopamine Dl-like receptor agonist clinically used as a vasodilator in cases of extreme hypertension (Murphy et al., 2001; Post and Frishman, 1998). Lithium has a much less defined function and is clinically used to treat a wide range of mental disorders, including bipolar disorder (Marmol, 2008). Furthermore, lithium is an inhibitor of GSK-3P and can have downstream effects on various signaling pathways including Wnt and Hedgehog. While lithium has been used to increase cilia length in a variety of cell types, other GSK-3P inhibitors have no effect on cilia length (Jope, 2003; Ou et al., 2009). This suggests that lithium has off- target effects beyond modulating cilium length. Thus only fenoldopam was studied further as a cilium lengthening agent.

Fenoldopam treatment increased cilia length, but also plays a role in adenylyl cyclase activity. The increase in forskolin stimulated adenylyl cyclase activity with fenoldopam treatment implicates two potential mechanisms. First, it is possible that fenoldopam sensitizes adenylyl cyclases, resulting in an increased cAMP response to forskolin. Alternatively, fenoldopam may increase production of adenylyl cyclases, augmenting forskolin stimulated cAMP production. This second notion is consistent with previous work indicating that fenoldopam treatment upregulates AC6, a specific adenylyl cyclase isoform, production in kidney cells (Yu et al., 2014). The results support this possible molecular pathway, demonstrating an increase in AC6 mRNA expression in response to fenoldopam stimulation. Previously, the inventors have demonstrated that AC6 localizes to the osteocyte primary cilium and is critical for primary cilia-mediated mechanotransduction (Kwon et al., 2010). Besschetnova et al, reported that stimulating the cAMP signaling pathway results in increased cilia length (Besschetnova et al., 2010). Using an siRNA mediated knockdown, they then showed that AC6 has a functional role in mediating primary cilium elongation.

Adenylyl cyclases and cAMP contribute to recovering and elongating primary cilia by stimulating IFT particle transport. It has previously been reported that stimulation of the adenylyl cyclase-cAMP-PKA signaling pathway augments anterograde transport of IFT particles to promote cilium elongation (Besschetnova et al., 2010). Because fenoldopam enhances adenylyl cyclase production, this suggests that fenoldopam treatment is potentiating adenylyl cyclase activity and IFT particle transport. The model of impaired cilia utilized an IFT88 knockdown, not a complete knockout of the gene, so it is possible that fenoldopam was able to enhance remaining IFT88 function and promote cilium elongation. Furthermore, this presupposes that even though the IFT88 knockdown is satisfactory to impair cilia formation and function, sufficient IFT88 remains to elongate cilia. The data show no change in IFT88 mRNA expression elicited by fenoldopam treatment suggesting that fenoldopam stimulated the remaining IFT88, rather than promoting production of new IFT88. This does not, however, discount the notion that fenoldopam treatment may instead prevent IFT88 knockdown driven disassembly of the cilium.

The specific means by which cells with longer cilia are more mechanosensitive remains elusive, but there are two potential mechanisms of how this may occur. Schwartz et al, developed one of the first models of primary cilia deflection under fluid flow and hypothesized that longer cilia would experience greater membrane strain to increase opening of stretch-activated ion channels on the ciliary membrane (Schwartz et al., 1997). Alternatively, longer cilia may simply allow for the presence of more cilia- specific proteins and signaling molecules within this microdomain (Breslow et al., 2013; Kee et al., 2012). Increasing the total amount of ciliary protein could enhance signal transduction within the ciliary compartment, modifying primary cilia-mediated mechanosensitivity. In fact, fenoldopam treated cells exposed to fluid flow have increased ciliary influx of calcium, which has been identified as one initiator of the mechanotransduction signaling cascade (Jin et al., 2014; Yuan et al., 2015). It is also possible that cilium-lengthening agents actually enhance ciliary protein production and trafficking to promote cilium elongation.

The correlation between cilia length and critical ciliary proteins involved in mechanosensing was examined with AC6 siRNA treatment. The knockdown of AC6 decreased flow-induced osteogenic gene expression, yet fenoldopam treatment was not sufficient to rescue AC6 expression or OPN expression as was demonstrated in the IFT88 knockdown model. Because fenoldopam treatment was not able to recover AC6 or OPN mPvNA expression in AC6 knockdown cells, this may suggest that the ability of fenoldopam to enhance AC6 activity is critical for recovering cellular mechanosensing. However, AC6 knockdown also decreased primary cilia length, which was not recovered with fenoldopam and did not alter cilia incidence. Altogether, these data suggest that both cilia length and protein production may be critical in primary cilia-mediated mechanosensing.

Cells with longer cilia are more mechanosensitive, but primary cilia cannot be elongated indefinitely. Longer cilia are exposed to greater drag force, and are more likely to be sheared off (Hierck et al., 2008). For example, endothelial cell primary cilia are flow sensors in regions of low shear specifically because they are cleaved off as shear stresses increase (Van der Heiden et al., 2008). Interestingly, electron microscopy has shown that primary cilia structure is not constant along the ciliary axoneme and becomes increasingly disorganized and asymmetric at the distal tip (Odor and Blandau, 1985; Yamamoto and Kataoka, 1986). This loss of microtubule symmetry reduces the bending stiffness of the cilium at the distal end, making drastically elongated cilia more susceptible to removal by fluid shear (Rydholm et al., 2010).

While osteocyte primary cilia are free-standing flow sensors in vitro, their mechanosensing function may differ in vivo. It has been estimated that the lacunar space in which osteocytes reside in vivo allows for only a 1 μπι long cilium (McNamara et al., 2009; Uzbekov et al., 2012). Due to the spatial limitations within the lacuna, the potential effect of pharmacologically enhancing osteocyte cilia length in vivo is unclear. In fact, these spatial constraints may point to the cilium not being a free-flowing mechanosensor at all. Rather, osteocyte cilia may anchor to the lacunar wall, similarly to chondrocyte primary cilia which form integrin attachments with the surrounding extracellular matrix, ECM (McGlashan et al., 2006). A computational model by Vaughan et al, simulated osteocytes exposed to fluid flow within the lacunar-canalicular network (Vaughan et al., 2014). The authors modeled a free-standing cilium, 0.5 μιη long, within a lacuna and calculated the resulting strain at the base of the cilium. Their model suggests that a cilium in this configuration does not experience a great enough strain to function as a flow sensor, but a cilium directly attached to the ECM does. The authors, however, do not account for the amount of membrane strain necessary to stimulate stretch-activated ion channels on the ciliary membrane, and may have overestimated the required strain for cilium stimulation. Fenoldopam treatment not only increases length, but may also enhance mechanosensitive protein levels, such as adenylyl cyclases and ion channels, within the cilium. Regardless of cilium length, this enriched protein trafficking to the cilium would increase chemical kinetics within the ciliary microdomain to modify cellular mechanosensitivity.

Targeting primary cilia-mediated mechanotransduction has widespread applications in preventative medicine that reach far beyond osteocytes. Numerous diseases are characterized by impaired primary cilia function. Mutations of PC2, polycystin 2, are attributed to polycystic kidney disease and skeletal deformations. Bardet-Biedl syndrome is characterized by malfunctioning BBS proteins at the base of the primary cilium, causing retinopathy, Polydactyly, and renal failure (Loktev et al., 2008; Mochizuki et al., 1996; Xiao et al., 2011). Recently, primary cilia have even been implicated in tumor development. Primary cilia help regulate Wnt signaling, changes in which have been correlated with cancer cell progression (Lancaster et al., 2011). Furthermore, some cancer cell types lose their primary cilia, which potentially contributes to their insensitivity to repressive signals (Plotnikova et al., 2008). Additionally, atherosclerotic plaques form in areas of low and disturbed arterial fluid flow, regions that interestingly have an increased incidence of primary cilia. This suggests that these cells are compensating, increasing their sensitivity to low fluid flow in order to promote an adequate cellular response (Van der Heiden et al., 2008; Warboys et al., 2011). Within bone, osteocytes utilize primary cilia to sense and respond to mechanical cues. In vitro and in vivo studies demonstrate that when these cilia are removed there is a decreased bone formation response to loading (Kwon et al., 2010; Malone et al., 2007; Temiyasathit et al., 2012). Fenoldopam is already an FDA approved drug, and The results point to it being an attractive candidate for study in numerous in vitro and in vivo applications to treat such a myriad of conditions. (7) References

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Yuan S, Zhao L, Brueckner M, Sun Z (2015) Intraciliary Calcium Oscillations Initiate Vertebrate Left-Right Asymmetry. Curr. Biol. 25: 556-567. doi: 10.1016/j .cub.2014.12.051.

Example 2: Primary cilia-mediated mechanotransduction regulates osteocyte paracrine signaling to osteoblasts

(1) Introduction

Primary cilia, solitary non-motile antennae, mediate mechanosensing in numerous cell types, such as kidney, cartilage, and bone cells [1, 2, 3]. Impairing/removing primary cilia diminishes cell mechanosensitivity [3]. Stiffening primary cilia, by increasing microtubule acetylation, decreases mechanosensitivity [4]. Paracrine signals from mechanically stimulated osteocytes promote MSC osteogenic differentiation [5]. Flow-induced mechanical stimulation initiates mechanotransduction [6]. Mechanical load is a potent anabolic stimulus of bone formation [3]. The objective of this study is to determine whether the osteocyte cilium is involved in signaling to osteoblasts and how this signaling can be potentiated via ciliary targeted therapies

(2) Methods

Cell Culture

MLO-Y4 osteocytes were cultured on collagen coated dishes. MC3T3 osteoblasts were cultured on tissue culture dishes. Osteocyte media was supplemented with 10 μΜ fenoldopam for 16 hours prior to rocking, 5 μΜ tubastatin for 3 hours, or DMSO vehicle control.

In Vitro Mechanical stimulation

Oscillatory fluid flow was applied to osteocytes at 0.12 Pa peak wall shear stress using a rocker plate set-up [8]. MLO-Y4s were subjected to 2, 6, 12, and 24 hours of flow. Cells not exposed to rocking were used as a no flow control. 12 hours of flow was then used for the remainder of studies. Osteocytes were subjected to drug treatment or siRNA-mediated knockdown prior to initiation of flow. Conditioned osteocyte media was used to culture osteoblasts for 24 hours before analysis.

In vivo drug treatment and loading

Skeletally mature, 16 week old, C57B1/6 mice were treated with 20 mg/kg fenoldopam by subcutaneous injection on 7 consecutive days. Compressive ulnar loading was applied on days 5-7 to mechanically stimulate bone (3N at 2 Hz for 120 cycles) [9].

Primary cilia disruption Osteocyte primary cilia formation was inhibited by siRNA-mediated knockdown of IFT88 [3, 5]. AC6, TRPV4, and PC2 were also inhibited by siRNA treatment [10].

Statistics

All data reported as mean ± SEM, *p < 0.05 **p < 0.01, ***p < 0.001.

(3) Results

Osteoblast osteogenic gene expression was enhanced by culture with conditioned media from mechanically stimulated osteocytes (Fig. 6). Osteopontin (OPN) expression was normalized to GAPDH and compared to no flow controls.

Osteocytes with longer primary cilia were more mechanosensitive. Fenoldopam treatment increased osteocyte primary cilia length, as well as the OPN and COX-2 response to flow. OPN and COX-2 expressions were normalized to GAPDH and compared to no flow controls (Fig. 7A-C). Osteoblast osteogenic gene expressions were diminished by disruption of osteocyte primary cilia formation (Fig. 7D). Primary cilia were impaired by siRNA-mediated KD of IFT88. Media from mechanically stimulated osteocytes with impaired cilia elicited an abrogated response in osteoblasts compared to scramble control.

Osteocyte primary cilia directed osteogenic paracrine signaling. Osteoblast osteogenic response to paracrine signals from mechanically stimulated osteocytes was assessed by osteopontin, OPN, mRNA expression (Fig. 8). Osteocytes were treated with fenoldopam (increase cilia length and mechanosensing), or tubastatin (increase cilia stiffness to impair mechanosensing). Osteocyte cilia formation was also inhibited, IFT88 knockdown, as well as pools of key cilia mechanotransduction proteins - AC6, PC2, TRPV4 (Fig. 8).

Fenoldopam treatment enhances load-induced bone formation (Fig. 9A).

Skeletally mature mice were administered fenoldopam for 7 consecutive days. Compressive ulnar load was applied for 3 days to mechanically stimulate the bones, while contralateral limbs served as non-loaded controls. Dynamic histomorphometric analysis was performed to asses bone adaptation. The amount of mineralizing surface (rMS/BS) remained unchanged, while mineral apposition (rMAR) and bone formation (rBFR/BS) rates significantly increased with fenoldopam treatment (Fig. 9A). N > 12 for each group. 2-way ANOVA revealed no statistical difference based on gender.

Load-induced bone formation was assessed by dynamic histo-morphometry (Fig. 9B). Alizarin (red) was administered four days after calcein (green). Bone formation was measured at periosteal surface. Minimal adverse effects of drug treatment is shown in Fig. 9C-E. There is no difference in visible bone ultrastructure between fenoldopam and vehicle control mice (Fig. 9C). Mouse weight, kidney weight, and kidney morphology assessed by H&E stain, remained unchanged in drug vs vehicle control (Fig. 9D-E). μCT analysis also revealed no change in normal bone properties due to drug treatment.

Table 1 : uCT analysis of ulnar midshaft shows no change in normal bone architecture with drug treatment compared to control.

Female Male

Bone and parameter Vehicle Fenoldopam Vehicle Fenoldopam

Ulnar midshaft

n 9 5 8 7

Total area (mm²) 0.352 ± 0.019 0.357 ± 0.005 0.380 ± 0.018 0.370 ± 0.027

Cortical area (mm²) 0.306 ± 0.017 0.310 ± 0.003 0.330 ± 0.016 0.320 ± 0.023

Cortical thickness

(mm) 0.173 ± 0.008 0.173 ± 0.006 0.172 ± 0.005 0.169 ± 0.009

\_max (mm⁴) 0.028 ± 0.004 0.029 ± 0.001 0.037 ± 0.006 0.033 ± 0.007 l_mm (mm⁴) 0.004 ± 0.001 0.005 ± 0.000 0.005 ± 0.001 0.004 ± 0.001

Porosity (%) 0.131 ± 0.005 0.131 ± 0.006 0.133 ± 0.004 0.134 ± 0.003

Bone Mineral Density 1236.399 ± 1224.143 ± 1234.686 ± 1218.543 ±

(mg/mm³) 15.089 11.750 25.702 20.338

(4) Discussion

Primary cilia disruption in mechanically stimulated osteocytes diminishes the osteogenic response in osteoblasts, demonstrating a role of primary cilia in osteocyte mechanotransduction, as well as downstream signaling to other cell types. Osteocytes are a paracrine signaling nexus that directs not only MSC differentiation, but also osteoblast activity Primary cilia disruption in osteocytes does not completely abolish the flow- induced OPN increase in osteoblasts, suggesting that other cellular mechanosensors, such as integrins or gap junctions, may also be involved. This paracrine signaling model does not discount the potential of intercellular signaling by cell-cell contact between mechanically stimulated osteocytes and osteoblasts. Pharmacologic manipulation of osteocyte cilia alters mechanotransduction response and paracrine signaling to osteoblasts.

Pharmacologically targeting the primary cilia apparatus can be a potential therapeutic strategy to promote bone formation. Treatment with a cilia lengthening agent can sensitize bone to mechanical stimulation. Increased mineral apposition and bone formation rates, with no change in amount of mineralizing surface, indicates increased osteoblast activity, consistent with in vitro results. Fenoldopam is a DR1 agonist clinically used to treat hypertension, but never used for any bone indication. No change in normal bone properties, animal weight, kidney weight, or kidney morphology suggests minimal adverse effects of drug treatment.

(5) References

1. Praetorius et al. (2003) JMembr Biol. 191(1) 69-76.

2. McGlashan et al. (2010) Cell Biol Int. 34(5):441-6.

3. Malone et a/. (2007) PNAS 104(33): 13325-13330.

4. Nguyen et al. (2015) Biol Open. 4(12): 1733-8.

5. Hoey et al. (2011) Biochem Biophys Res Commun. 412(1): 182-7.

6. Battle et al. (2015) PNAS 112(5): 1410-5.

7. Duffy and Jacobs (2015) Biophys J 108(7): 1583-4.

8. Zhou et al. (2010) JBiomech. 43(8): 1598-602.

9. Lee et al. (2014) FASEBJ. 28(3): 1 157-65. [10] Lee et al. (2015) Cilia. 4:7.

Example 3: Targeting the mechanobiology of the osteocyte primary cilium to bias bone formation Introduction

The burden of osteoporosis and low bone mass is unrelenting, affecting over 50% of the US population over 50, and is compounded by the insufficiency of prophylaxis and treatment options. The inventors and others have established the osteocyte primary cilium - a mechanosensing antenna-like organelle - as a promising pharmaceutical target to exploit the natural anabolic response to physical loading to maintain bone health. Nonetheless, how key molecular components of the osteocyte primary cilium microdomain can be manipulated to enhance bone formation without adversely impacting bone physiology remains a critical gap in knowledge. The central hypothesis is that the unique mechanobiology of the osteocyte primary cilium can be targeted to bias bone formation while minimizing disruption to normal physiology. This is based on the rationale that the osteocyte primary cilium plays a key role in bone mechanotransduction and preliminary data suggest it can be selectively manipulated. Specifically agents that modify cilium structural properties (length and stiffness) and its osteogenic signaling modulate load-induced bone formation. Furthermore, preliminary evidence collected with the inventors' novel biosensors indicates that aspects of calcium/cAMP signaling dynamics within the ciliary microdomain are unique to osteocytes. Particularly, with a ciliary-localizing calcium biosensor, the inventors have identified TRPV4 as the principal mechanosensitive calcium ion channel. The inventors have also demonstrated that AC6 is an important adenylyl cyclase in osteocyte primary cilia and that mice lacking AC6 have an abrogated response to load-induced bone formation, similar to an osteocyte primary cilia knockout model. With the expanding biosensor capabilities, the inventors will define distinct molecular targets of the osteocyte cilium and employ a multifaceted approach designed to ensure that the proposed therapeutic strategy does not have off-target effects on intercellular signaling or remodeling.

The following specific aims will test the central hypothesis by establishing the potential of osteocyte cilia therapeutics in vivo (SA1), defining the osteocyte cilia microdomain signaling cascade to inform development of improved treatment strategies (SA2), and characterize the role of the osteocyte primary cilium in directing bone intercellular communication and physiology (SA3).

SA1: Therapeutically manipulate the physical and biochemical composition of the primary cilium microdomain and quantify the resulting change in load- induced bone formation in vivo.

Working hypothesis: Biochemically potentiating the ciliary microdomain increases load-induced bone formation. Skeletally mature mice will be treated with agents that lengthen (fenoldopam) and stiffen (tubastatin) primary cilia. TRPV4 will be agonized (4aPDD, GSK101) and antagonized (RN1734). Load-induced bone formation will be quantified via dynamic histomorphometry, microCT analysis, and mechanical testing.

SA2: Characterize the osteocyte intraciliary molecular signaling apparatus and determine how its dynamics are affected by altering ciliary structure and composition. Working Hypothesis: Calcium kinetics and cAMP signaling are coupled by adenylyl cyclases within the cilium and changes in their dynamics culminate in altered osteogenic gene expression. This aim relies on unique ciliary cAMP and calcium biosensors to measure calcium and cAMP within the ciliary microdomain. Coupling between these two second messengers will be confirmed by introducing a disruptive mutation to the AC6 calcium binding pocket. Targeting sequences will be altered to manipulate ciliary pools of adenylyl cyclases and channel proteins to enhance mechanosensitivity. TRPV4-calcium with a channel-biosensor fusion construct will be observed to distinguish ciliary from cytoplasmic influx. Finally, the inventors will determine whether mechanically induced ciliary cAMP synthesis occurs and distinguish the temporal relationship between ciliary and cytosolic cAMP using the ciliary-direct cAMP biosensor.

SA3: Elucidate the role of osteocyte primary cilia in the propagation of signals through the osteocyte network and downstream regulation of bone turnover.

Working Hypothesis: Manipulating primary cilia sensitivity biases osteocytes towards osteogenic signaling while maintaining osteocyte function in activating bone cells and transmitting intercellular signals. Ciliary knockouts and pharmacologically enhanced primary cilia will be tested in terms of four aspects of bone physiology: (1) inter-osteocyte signaling with an ex vivo load-induced calcium signal propagation model; (2) osteocyte-osteoblast signaling in a co-culture model; (3) fatigue-induced osteoclastogenesis; and (4) disuse bone loss and recovery in a transient paralysis model. The expected outcomes are in vivo evidence of the potential to sensitize osteocyte primary cilia (SA1), identification of osteocyte interciliary signaling mechanisms to inform development of new therapeutic strategies (SA2) and determination of the impact of targeting osteocyte primary cilia on anabolic and catabolic signaling (SA3). This will have a positive impact to advance bone mechanobiology and contribute to the development of a more complete armamentarium to treat osteoporosis.

Results

SA1: Therapeutic manipulation of the physical and biochemical composition of the primary cilium microdomain and quantification of the resulting change in load-induced bone formation in vivo. Introduction: Previously the focus was to further the understanding of how primary cilia-mediated mechanotransduction allows osteocytes to sense and respond to mechanical cues. The inventors demonstrated that the primary cilium plays a critical role in osteocyte mechanobiology and that key structural and molecular aspects of this unique microdomain regulate its function in vitro. The objective of this aim is to determine if the effects of agents shown to alter primary cilia mechanosensitivity in vitro can be translated in vivo to enhance bone adaptation. It will be achieved by testing the working hypothesis that biochemical potentiation of the ciliary microdomain increases load- induced bone formation. The approach will be to target primary cilia structure and mechanosensing proteins via subcutaneous injections of pharmacologic agents in mice. Primary cilia stiffness will be modulated by tubastatin treatment, length will be targeted with fenoldopam, and the TRPV4 ion channel will be agonized with 4aPDD and GSK1019670A and antagonized with RN-1734. Mouse long bones will then be subjected to compressive loading to induce bone formation and the bone adaptation response will be assessed by dynamic histomorphometry. The expectation is that the inventors will show that treatment with primary cilia manipulating agents can alter the bone response to loading, indicating the primary cilium as not only a potential, but a viable and promising target for bone disease therapeutics. Furthermore, this will demonstrate an in vitro to in vivo scheme for the development of primary cilia targeted interventions for conditions such as osteoporosis.

Preliminary Studies: Skeletally mature C57BL/6J mice (16 weeks old) were administered subcutaneous injections of 4aPDD, fenoldopam, tubastatin A, or vehicle control and axial compressive ulnar loading followed by dynamic histomorphometry (Fig 10). Results were quantified as the loaded limb relative to the non-loaded contralateral limb. The initial dose of 4aPDD, in only 3 animals, did not promote a significant increase in relative bone formation rate compared to vehicle control (rBFR/BS). A dose- response study was done for fenoldopam and there was a marked increase in response from 2 mg/kg to 20 mg/kg. Tubastatin treatment slightly decreased rBFR, and also resulted in an over 50% decrease (data not shown) in active mineralizing surface (rMS/BS). This effect was dramatic enough to warrant continued in vivo evaluation. Additionally, gross changes in size, weight, or temperament as a result of the injections was not observed. For fenoldopam (20 mg/kg) treated specimens, kidney histology showed no abnormalities or cysts and weight to body mass measurements were no different than vehicle controls. In addition, μCT analysis of these mice demonstrated no change in normal cortical bone properties including thickness, area, and moment of inertia (data not shown). This suggested that the treatments are well tolerated with no observed no unintended adverse consequences. These data support the working hypothesis that biochemically potentiating the ciliary microdomain increases load- induced bone formation, and that all of the required techniques to complete the aim are up-and-running in the inventors' hands.

SA2: Characterization of the osteocyte intraciliary molecular signaling apparatus and determination how its dynamics are affected by altering ciliary structure and composition.

Introduction: While SA1 focused on immediate translation of pharmacological agents to enhance bone formation, SA2 focused on further elucidating the osteocyte cilia signaling microdomain to enable development of highly specific therapeutic strategies. Previously, the inventors identified a unique signaling mechanism of the primary cilium involving calcium influx through TRPV4 and cAMP production by AC6. Both, TRPV4 and AC6, have independently been shown to be important for mechanically-induced osteogenic signaling, though it is not known if they work in concert in the ciliary microdomain to regulate calcium/cAMP dynamics and downstream osteogenic signaling. A mechanistic understanding of osteocyte intraciliary signaling dynamics is impeding the refinement of molecular targets. Due to the amplifying nature of their signaling cascades, minute alterations in second messengers have large downstream impacts and strategies targeting them have promising pharmacological potency. Furthermore, there is great potential for therapeutic specificity in promoting bone growth since the osteocyte signaling apparatus is distinct from other cell types. The objective is to determine how osteocyte primary cilia microdomain potentiates signaling in response to mechanical stimulation. To achieve this objective the inventors will test the working hypothesis that calcium kinetics and cAMP signaling are coupled by adenylyl cyclases within the cilium and changes in their dynamics culminate in altered osteogenic gene expression. The approach is to utilize molecular techniques to characterize the interplay of intraciliary calcium/cAMP signaling. Specifically, novel biosensors will be utilized to quantify changes in calcium/cAMP signaling with alterations in axonemal stiffness, cilium length, and the presence and concentration of ciliary proteins believed to be involved in mechanotransduction. The hypothesis is that the composition and function of the osteocyte ciliary microdomain can be altered to enhance the calcium/cAMP signaling apparatus, thereby activating osteocytes independently of other cell types to encourage bone growth.

Preliminary Studies: A mechanism was developed to detect ciliary cAMP levels specifically by fusing a FRET-based Epacl-cAMP biosensor to Arll3b, a protein that is naturally trafficked exclusively to the primary cilium. With shear flow, a ciliary cAMP increase was observed approximately 20 seconds following a spike in calcium (Fig. 11), suggesting the latter induced the former. The cytosolic cAMP levels decreased approximately 30 seconds following an increase in ciliary cAMP (data not shown). These results demonstrate that the ciliary and cytosolic signaling domains are distinct and it is feasible to study their dynamics using the novel biosensors.

To determine if AC6 couples calcium and cAMP in the osteocyte cilium, an overexpression vector was generated by inserting AC6 into a pcDNA3.2 backbone

382 426

(pcDNA3.2+AC6). Two AC6 aspartic acid residues, Asp and Asp , were mutated via site directed mutagenesis to disrupt the calcium binding pocket (pcDNA3.2+AC6^CalMut). This mutation did not affect AC6's general catalytic activity by treating transfected cells with 10 μΜ of forskolin (which binds at an independent site) for 20 minutes and verified that cAMP production was not affected by the mutation (data not shown).

To identify AC isoforms other than AC6 that may mediate calcium/cAMP coupling, and ICC revealed that AC3 localizes to osteocyte but not kidney cilia (Fig. 14). This is the first examination of ciliary AC3 expression in non-excitable cells and is compelling evidence that there are unique components of the osteocyte cilium. To determine if calcium and cAMP are coupled and how calcium regulates inter-ciliary protein activity, plasmids of TRPV4, AC6 and AC3 were acquired and constructed. All plasmids were transferred into a common backbone, pcDNA3.2, to generate overexpression vectors. With site directed mutagenesis, the calmodulin binding domain of TRPV4 (Δ812-831) were deleted, and the calcium binding pocket of AC6 (Asp³⁸² and Asp⁴²⁶ to Ala) and AC3 (Asp³²⁴ and Asp³⁶⁸ to Ala) were mutated. TRPV4 mutations were previously validated. The results confirmed AC mutations did not affect protein activity by treating transfected cells with 10 μΜ of forskolin (which binds at an independent site) for 20 minutes and verified that cAMP production was not affected by the mutation (data not shown). Mutating TRPV4 and AC6 results in a decreased osteogenic response to flow compared to the wildtype overexpression vector, while mutated AC3 increased the osteogenic response to flow. These data demonstrate that calcium is involved in regulating osteogenic signaling through adenylyl cyclases and that all plasmids are in-hand and can be successfully transfected..

With flow, the mutant group had higher levels of cAMP (Fig. 12 A), demonstrating that calcium inhibition was lost as expected and AC6 overexpression resulted in an enhanced osteogenic response that was lost in the mutants (Fig. 12A).

Next a mutant plasmid (pcDNA3.2+AC6^VxPMut) was generated to prevent trafficking of AC6 to the cilium. The inventors mutated two residues of a potential ciliary targeting sequence, a VxP motif, on the first intracellular loop. Indeed, AC6 overexpression in the cilium was lost with this mutation (Fig. 13). These results show that trafficking sequences can be genetically altered to selectively direct or prevent localization of proteins to influence osteogenesis.

Finally, the calcium biosensor was linked to a TRPV4-YFP plasmid (gift from Heinrich Brinkmeier, University of Greifswald) to measure calcium entry independently from other calcium sources (Fig. 17). This same strategy will be employed to traffic TRPV4 exclusively to the cilium with Aril 3b. To summarize the plasmid-based data, all sequences are in-hand and have been developed to varying degrees to validate the proposed experimental approach.

Collectively, the data suggest the osteocyte microdomain contains potentially unique proteins with specific functional domains that may become compelling targets for enhancing osteogenesis. They also suggest that a detailed understanding of ciliary calcium/cAMP dynamics will reveal new therapeutic candidates and that the molecular strategies to prosecute this aim are validated.

SA3: Elucidation of the role of osteocyte primary cilia in the propagation of signals through the osteocyte network and downstream regulation of bone turnover.

Introduction: With a long-term goal of therapeutically targeting the osteocyte primary cilium, it is critical to overcome the limitations of current osteoporosis treatments to only promote formation or inhibit resorption. For instance, bisphosphonates increase bone mass, but can lead increased microdamage accumulation eventually resulting in atypical femur fractures. While there is an emerging understanding of the role of osteocyte primary cilia in mechanosensing at the cellular and tissue level, it is crucial to future therapeutics to also understand osteocyte ciliary contribution to normal intercellular communication. The objective is to determine the role of osteocyte primary cilia in intercellular communication and if osteocyte mechanosensitivity can be manipulated while otherwise maintaining normal bone biology. This objective is pursued by testing the working hypothesis that the role of osteocytes in activating bone cells and transmitting intercellular signals is unchanged while manipulating their primary cilia. To that end, the approach examines three key aspects of bone biology: osteocyte intercellular communication, osteogenic signaling from osteocytes to osteoblasts and MSCs, and osteoclastogenesis. In Study 3.1 the impact on intracellular signaling in the model of the intact osteocyte network will be examined. Osteoclast activity due to disuse and overuse may occur through different mechanisms. For example overuse may produce microdamage, which could recruit osteoclasts independent of primary cilia. To address this, two independent models will be used: a newly developed repetitive loading model and the transient muscle-paralysis model. Additionally, as muscle function is regained in the paralysis model, normal bone mass is recovered, which allows us to examine load-induced bone formation with restoration of normal ambulation. Paracrine signaling between treated osteocytes and osteoblasts/MSCs will be examined to assess the effects on osteogenic signaling. The expectation is that altering osteocyte cilium mechanics will affect initial calcium signaling, promote osteogenic signaling, and allow normal osteoclast activation. These data would indicate that osteocyte ciliary mechanosensitivity can be targeted to shift the level of osteogenic signaling, while protecting normal bone rejuvenation.

Preliminary Data: Whether manipulations of primary cilia have adverse consequences for osteocyte network response will be examined using a novel ex vivo model the inventors developed that tracks real-time intracellular calcium levels in an intact osteocyte network. An initial study was performed to validate the system and verify the osteocyte network response to physical loading (Fig. 15).

Osteoclastogenesis will be investigated using two potent stimuli: overloading and disuse. RA KL/OPG is a common readout for osteocytic regulation of osteoclastogenesis. For this study, a repetitive loading model of osteoclastogenesis was developed, crucial to enable exploitation of the extensive genetic mouse models. In a pilot experiment mice were subjected to a relatively small amount of cyclic overloading (roughly 1000 cycles). The mice responded to higher strains (3000

and cycle number (-1000) than the model of habitual loading used in SA1 with a dramatically increased RANKL/OPG mRNA ratios (Fig. 16), demonstrating that this model to study osteoclast activation, is up-and-running. The impact of potentiating osteocyte primary cilia on osteocyte-osteoblast communication will be evaluated in a co-culture system. In a pilot study the inventors treated osteocytes with fenoldopam (lengthening agent) and tubastatin A (stiffening agent) to enhance or diminish mechanosensitivity, respectively. Manipulating osteocyte primary cilia affected paracrine signaling to osteoblasts and that it was achievable to both promote (fendoldopam) and diminish (tubastatin A) osteogenic response in osteoblasts. As shown in Fig. 8 (left panel) MLO-Y4 cells were seeded on a T75 flask. Fenoldopam (10 μΜ) was added 16 hours prior, while tubastatin A (5 μΜ) was added 4 hours before stimulation. Flasks with fresh media were placed in an incubator and rocked at 0.5 Hz with an amplitude of 1.5 cm for 12 hrs. Controls were placed in the same incubator, but not rocked. 12 mL of media from each flask was transferred to a 4-well plate of MC3T3- El osteoblasts. After 24 hours, increased osteogenic gene expression was observed with lengthening (fenoldopam) and decreased with stiffening (tubastatin) Flow over no-flow control data were used as comparison (n=4, ***p < .001, Mean ± SEM).

In summary the results suggest osteocyte cilia mechanosensing can be modulated to increase or decrease osteogenic activity. Furthermore the inventors have expanded the experimental capabilities in order to evaluate potential adverse effects of cilia targeted therapies on key aspects of bone biology.

Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, and composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the presently disclosed subject matter, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the presently disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps. Patents, patent applications, publications, product descriptions and protocols are cited throughout this application the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

CLAIMS We claim:

1. A method of increasing expression of a gene in a cell comprising:

contacting the cell with an effective amount of a cilium elongation modulator, wherein the cilium elongation modulator increases a length of one or more primary cilia of the cell.

2. The method of claim 1, wherein the cilium elongation modulator is contacted to the cell in an amount effective to increase mechanosensitivity of the cell.

3. The method of claim 1, wherein the cilium elongation modulator is contacted to the cell in an amount effective to increase cAMP level in the cell.

4. The method of claim 3, wherein the cilium elongation modulator is contacted to the cell in an amount effective to increase expression level or an enzymatic activity of adenylyl cyclase.

5. The method of claim 1, wherein the cilium elongation modulator comprises a dopamine Dl -like receptor agonist, derivatives thereof, and combinations thereof.

6. The method of claim 5, wherein the cilium elongation modulator is selected from the group consisting of fenoldopam, Dihydrexidine (CAS No: 158704-02-0), Dopamine (CAS No:62-31-7), NPEC-caged-dopamine (CAS No: 1257326-23-0), SKF 38393 (CAS No:20012-10-6), SKF 77434 (CAS No:300561-58-4), SKF 81297 (CAS No:67287-39- 2), SKF 82958 (CAS No:74115-01-8), SKF 83822 (CAS No:74115-10-9), SCH-23390 (CAS No: 87075-17-0), SKF-83959 (CAS No: 67287-95-0), A68930 (CAS No: 130465- 39-3), A77636 (CAS No: 145307-34-2), (R)-(-)-Apomorphine (CAS No: 314-19-2), CY 208-243 (CAS No: 100999-26-6), Ecopipam (SCH-39, 166, CAS No: 112108-01-7), derivatives thereof, and combinations thereof.

7. The method of claim 5, wherein the dopamine Dl-like receptor agonist is selected from the group consisting of fenoldopam, derivatives thereof, and combinations thereof.

8. The method of claim 1, wherein the cilium elongation modulator is selected from the group consisting of lithium, derivatives thereof, and combinations thereof.

9. The method of claim 1, wherein the cilium elongation modulator comprises an adenylyl cyclase agonist, derivatives thereof, and combinations thereof.

10. The method of claim 9, wherein the cilium elongation modulator is selected from the group consisting of fenoldopam, forskolin, NKH 477 (CAS No: 138605-00-2), PACAP 1-27 (CAS No: 127317-03-7), PACAP 1-38 (137061-48-4), derivatives thereof, and combinations thereof.

11. The method of claim 1, wherein the cillium elongation modulator increases the expression of one or more gene selected from the group consisting of AKT1, BBS4, CCND1, CDK5RAP2, CDKN1A (P21CIP1/WAF1), IGF1, INS2, MAP2K1, PKD1, PKD2, TRP53. DYNC2LI1, IFT172, IFT20, IFT74, IFT80, TRPV4, IFT88, Kinesin- like protein (KIF3A, KTF3B), ALMSl, ARL6, BBS1, BBS2, BBS4, BBS7, IFT172, IFT88, MKKS, OFD1, PKHD1, RPGRIP1L, VANGL2 and WWTR1.

12. The method of claim 1, wherein the cell is an osteocyte, an osteoblast, an osteoclast, an osteoprogenitor cell, or a combination thereof.

13. The method of claim 1, wherein the cillium elongation modulator is contacted to a population of cells.

14. The method of claim 1, wherein the gene is an osteogenic gene.

15. The method of claim 14, wherein the osteogenic gene is selected from the group consisting of COX-2, OPN, BSP, Collagen I, Osteocalcin, Runt-related transcription factor 2 (Runx2, (Cbfal/PEBP2aA/AML-3/Osf2)), Osterix (Osx), distal-less homeobox protein 5 (Dlx5), Alkaline phosphatase (ALP), Msx-2 (Hox-8), Nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κΒ), Osteoprotegerin (OPG), Cytochrome c oxidase subunit 2 (Cox-2), fibroblast growth factor 2 (FGF2), bagpipe homeobox homolog 1 (Drosophila) (Bapxl), Collagen I, Osteocalcin, Osteopontin (OPN), Bone sialoprotein (BSP), AHSG, AMBN, AMELY, BGLAP, ENAM, MINPP1, STATH, TUFTl, Cartilage condensation genes: BMPl, COL11A1, SOX9, ALPL, AMBN, AMELY, BGLAP, CALCR, CDHl l, DMP1, DSPP, ENAM, MINPP1, PHEX, RUNX2, STATH, TFIPl l, TUFTl, ANXA5, BGLAP, BMPl, CALCR, CDHl l, COMP, DMP1, EGF, MMP2, MMP8, VDR, BMPl, BMP2, BMP3, BMP4, BMP 5, BMP6, CSF2, CSF3, EGF, EGFR, FGFl, FGF2, FGF3, FGFRl, FGFR2, FLTl, GDF10, IGF1, IGF1R, IGF2, PDGFA, TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, VEGFA, VEGFB, COL4A3, COL10A1, COL11A1, COL12A1, COL14A1, COL15A1, COL1A1, COL1A2, COL2A1, COL3A1, COL4A3, COL5A1, AHSG, COL4A3, SERPINHl, CTSK, MMP10, MMP2, MMP8, MMP9, and PHEX, CDHl l, COL11A1, COL14A1, ICAM1, ITGB 1, VCAM1, ITGA1, ITGA2, ITGA3, ITGAM, ITGB 1, BGLAP, CD36, COL12A1, COL15A1, COL4A3, COL5A1, COMP, FN1, SCARB1, TNF, MSX1, NFKBl, RUNX2, SMADl, SMAD2, SMAD3, SMAD4, SOX9, TNF, TWIST 1, VDR, and combinations thereof.

16. The method of claim 15, wherein the osteogenic gene is selected from the group consisting of COX-2, OPN, BSP, Collagen I, Osteocalcin, and combinations thereof.

17. The method of claim 14, wherein the cell is comprised in a population of cells, and the cilium elongation modulator is contacted to the population of cells in an amount effective to increase osteogenesis.

18. The method of claim 1, wherein the cell is a mammalian cell.

19. The method of claim 18, wherein the mammalian cell is a human cell.

20. A method of treating a ciliopathy in a subject comprising:

administering to a subject in need thereof, an effective amount of a cilium elongation modulator,

wherein the cilium elongation modulator increases a length of one or more primary cilia of a cell in the subject.

21. The method of claim 20, wherein the cilium elongation modulator is administered to the subject in an amount effective to increase expression of a gene in the cell of the subject.

22. The method of claim 20, wherein the cilium elongation modulator is administered to the subject in an amount effective to increase mechanosensitivity of the cell of the subject.

23. The method of claim 20, wherein the cilium elongation modulator is contacted to the cell in an amount effective to increase cAMP level in the cell.

24. The method of claim 23, wherein the cilium elongation modulator is contacted to the cell in an amount effective to increase expression level or an enzymatic activity of adenylyl cyclase.

25. The method of claim 20, wherein the cilium elongation modulator comprises a dopamine Dl -like receptor agonist, derivatives thereof, and combinations thereof.

26. The method of claim 20, wherein the cilium elongation modulator is selected from the group consisting of fenoldopam, Dihydrexidine (CAS No: 158704-02-0), Dopamine (CAS No:62-31-7), NPEC-caged-dopamine (CAS No: 1257326-23-0), SKF 38393 (CAS No:20012-10-6), SKF 77434 (CAS No:300561-58-4), SKF 81297 (CAS No:67287-39-2), SKF 82958 (CAS No:74115-01-8), SKF 83822 (CAS No:74115-10-9), SCH-23390 (CAS No: 87075-17-0), SKF-83959 (CAS No: 67287-95-0), A68930 (CAS No: 130465-39-3), A77636 (CAS No: 145307-34-2), (R)-(-)-Apomorphine (CAS No: 314-19-2), CY 208-243 (CAS No: 100999-26-6), Ecopipam (SCH-39,166, CAS No: 112108-01-7), derivatives thereof, and combinations thereof.

27. The method of claim 26, wherein the dopamine Dl-like receptor agonist is selected from the group consisting of fenoldopam, derivatives thereof, and combinations thereof.

28. The method of claim 20, wherein the cilium elongation modulator is selected from the group consisting of lithium, derivatives thereof, and combinations thereof.

29. The method of claim 20, wherein the cilium elongation modulator comprises an adenylyl cyclase agonist, derivatives thereof, and combinations thereof.

30. The method of claim 29, wherein the cilium elongation modulator is selected from the group consisting of fenoldopam, forskolin, NKH 477 (CAS No: 138605-00-2), PACAP 1-27 (CAS No: 127317-03-7), PACAP 1-38 (137061-48-4), derivatives thereof, and combinations thereof.

31. The method of claim 20, wherein the cillium elongation modulator increases the expression of one or more gene selected from the group consisting of AKT1, BBS4, CCND1, CDK5RAP2, CDKN1A (P21CIP1/WAF1), IGF1, INS2, MAP2K1, PKD1, PKD2, TRP53. DYNC2LI1, IFT172, IFT20, IFT74, IFT80, TRPV4, IFT88, Kinesin- like protein (KIF3A, KTF3B), ALMSl, ARL6, BBS1, BBS2, BBS4, BBS7, IFT172, IFT88, MKKS, OFD1, PKHD1, RPGRIP1L, VANGL2 and WWTR1.

32. The method of claim 20, wherein the cell is an osteocyte, an osteoblast, an osteoclast, an osteoprogenitor cell, or a combination thereof.

33. The method of claim 20, wherein the gene is an osteogenic gene.

34. The method of claim 33, wherein the osteogenic gene is selected from the group consisting of COX-2, OPN, BSP, Collagen I, Osteocalcin, Runt-related transcription factor 2 (Runx2, (Cbfal/PEBP2aA/AML-3/Osf2)), Osterix (Osx), distal-less homeobox protein 5 (Dlx5), Alkaline phosphatase (ALP), Msx-2 (Hox-8), Nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κΒ), Osteoprotegerin (OPG), Cytochrome c oxidase subunit 2 (Cox-2), fibroblast growth factor 2 (FGF2), bagpipe homeobox homolog 1 (Drosophila) (Bapxl), Collagen I, Osteocalcin, Osteopontin (OPN), Bone sialoprotein (BSP), AHSG, AMBN, AMELY, BGLAP, ENAM, MINPPl, STATH, TUFTl, Cartilage condensation genes: BMPl, COL11A1, SOX9, ALPL, AMBN, AMELY, BGLAP, CALCR, CDHl l, DMP1, DSPP, ENAM, MINPPl, PHEX, RUNX2, STATH, TFIPl l, TUFTl, ANXA5, BGLAP, BMPl, CALCR, CDHl l, COMP, DMP1, EGF, MMP2, MMP8, VDR, BMPl, BMP2, BMP3, BMP4, BMP 5, BMP6, CSF2, CSF3, EGF, EGFR, FGFl, FGF2, FGF3, FGFRl, FGFR2, FLTl, GDF10, IGF1, IGF1R, IGF2, PDGFA, TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, VEGFA, VEGFB, COL4A3, COL10A1, COL11A1, COL12A1, COL14A1, COL15A1, COL1A1, COL1A2, COL2A1, COL3A1, COL4A3, COL5A1, AHSG, COL4A3, SERPINH1, CTSK, MMP10, MMP2, MMP8, MMP9, and PHEX, CDH11, COL11A1, COL14A1, ICAM1, ITGB 1, VCAM1, ITGA1, ITGA2, ITGA3, ITGAM, ITGB 1, BGLAP, CD36, COL12A1, COL15A1, COL4A3, COL5A1, COMP, FN1, SCARB1, TNF, MSX1, NFKB1, RUNX2, SMAD1, SMAD2, SMAD3, SMAD4, SOX9, TNF, TWIST 1, VDR, and combinations thereof.

35. The method of claim 34, wherein the osteogenic gene is selected from the group consisting of COX-2, OPN, BSP, Collagen I, Osteocalcin, and combinations thereof.

36. The method of claim 34, wherein the cell is comprised in a population of cells, and the cilium elongation modulator is contacted to the population of cells in an amount effective to increase osteogenesis.

37. A method of treating osteoporosis in a subject comprising:

38. The method of claim 37, wherein the cilium elongation modulator is administered to the subject in an amount effective to increase expression of a gene in the cell of the subject.

39. The method of claim 37, wherein the cilium elongation modulator is administered to the subject in an amount effective to increase mechanosensitivity of the cell of the subject.

40. The method of claim 37, wherein the cilium elongation modulator is contacted to the cell in an amount effective to increase cAMP level in the cell.

41. The method of claim 40, wherein the cilium elongation modulator is contacted to the cell in an amount effective to increase expression level or an enzymatic activity of adenylyl cyclase.

42. The method of claim 37, wherein the cilium elongation modulator comprises a dopamine Dl -like receptor agonist, derivatives thereof, and combinations thereof.

43. The method of claim 37, wherein the cilium elongation modulator is selected from the group consisting of fenoldopam, Dihydrexidine (CAS No: 158704-02-0), Dopamine (CAS No:62-31-7), NPEC-caged-dopamine (CAS No: 1257326-23-0), SKF 38393 (CAS No:20012-10-6), SKF 77434 (CAS No:300561-58-4), SKF 81297 (CAS No:67287-39-2), SKF 82958 (CAS No:74115-01-8), SKF 83822 (CAS No:74115-10-9), SCH-23390 (CAS No: 87075-17-0), SKF-83959 (CAS No: 67287-95-0), A68930 (CAS No: 130465-39-3), A77636 (CAS No: 145307-34-2), (R)-(-)-Apomorphine (CAS No: 314-19-2), CY 208-243 (CAS No: 100999-26-6), Ecopipam (SCH-39,166, CAS No: 112108-01-7), derivatives thereof, and combinations thereof.

44. The method of claim 42, wherein the dopamine Dl-like receptor agonist is selected from the group consisting of fenoldopam, derivatives thereof, and combinations thereof.

45. The method of claim 37, wherein the cilium elongation modulator is selected from the group consisting of lithium, derivatives thereof, and combinations thereof.

46. The method of claim 37, wherein the cilium elongation modulator comprises an adenylyl cyclase agonist, derivatives thereof, and combinations thereof.

47. The method of claim 46, wherein the cilium elongation modulator is selected from the group consisting of fenoldopam, forskolin, NKH 477 (CAS No: 138605-00-2), PACAP 1-27 (CAS No: 127317-03-7), PACAP 1-38 (137061-48-4), derivatives thereof, and combinations thereof.

48. The method of claim 37, wherein the cillium elongation modulator increases the expression of one or more gene selected from the group consisting of AKT1, BBS4, CCND1, CDK5RAP2, CDKN1A (P21CIP1/WAF1), IGF1, INS2, MAP2K1, PKD1, PKD2, TRP53. DYNC2LI1, IFT172, IFT20, IFT74, IFT80, TRPV4, IFT88, Kinesin- like protein (KIF3A, KIF3B), ALMSl, ARL6, BBS1, BBS2, BBS4, BBS7, IFT172, IFT88, MKKS, OFD1, PKHD1, RPGRIP1L, VANGL2 and WWTR1.

49. The method of claim 37, wherein the gene is an osteogenic gene.

50. The method of claim 49, wherein the osteogenic gene is selected from the group consisting of COX-2, OPN, BSP, Collagen I, Osteocalcin, Runt-related transcription factor 2 (Runx2, (Cbfal/PEBP2aA/AML-3/Osf2)), Osterix (Osx), distal-less homeobox protein 5 (Dlx5), Alkaline phosphatase (ALP), Msx-2 (Hox-8), Nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κΒ), Osteoprotegerin (OPG), Cytochrome c oxidase subunit 2 (Cox-2), fibroblast growth factor 2 (FGF2), bagpipe homeobox homolog 1 (Drosophila) (Bapxl), Collagen I, Osteocalcin, Osteopontin (OPN), Bone sialoprotein (BSP), AHSG, AMBN, AMELY, BGLAP, ENAM, MINPPl, STATH, TUFTl, Cartilage condensation genes: BMPl, COL11A1, SOX9, ALPL, AMBN, AMELY, BGLAP, CALCR, CDHl l, DMP1, DSPP, ENAM, MINPPl, PHEX, RUNX2, STATH, TFIPl l, TUFTl, ANXA5, BGLAP, BMPl, CALCR, CDHl l, COMP, DMP1, EGF, MMP2, MMP8, VDR, BMP1, BMP2, BMP3, BMP4, BMP 5, BMP6, CSF2, CSF3, EGF, EGFR, FGFl, FGF2, FGF3, FGFRl, FGFR2, FLTl, GDF10, IGF1, IGF1R, IGF2, PDGFA, TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, VEGFA, VEGFB, COL4A3, COL10A1, COL11A1, COL12A1, COL14A1, COL15A1, COL1A1, COL1A2, COL2A1, COL3A1, COL4A3, COL5A1, AHSG, COL4A3, SERPINH1, CTSK, MMP10, MMP2, MMP8, MMP9, and PHEX, CDH11, COL11A1, COL14A1, ICAM1, ITGB 1, VCAM1, ITGA1, ITGA2, ITGA3, ITGAM, ITGB 1, BGLAP, CD36, COL12A1, COL15A1, COL4A3, COL5A1, COMP, FN1, SCARB1, TNF, MSX1, NFKBl, RUNX2, SMADl, SMAD2, SMAD3, SMAD4, SOX9, TNF, TWIST 1, VDR, and combinations thereof.

51. The method of claim 52, wherein the osteogenic gene is selected from the group consisting of COX-2, OPN, BSP, Collagen I, Osteocalcin, and combinations thereof.

52. The method of claim 52, wherein the cell is comprised in a population of cells, and the cilium elongation modulator is contacted to the population of cells in an amount effective to increase osteogenesis.