AU2015201033B2 - Activin-ActRIIa antagonists and uses for promoting bone growth - Google Patents

Abstract

ACTIVIN-ACTRIIA ANTAGONISTS AND USES FOR PROMOTING BONE GROWTH In certain aspects, the present invention provides compositions and methods for promoting bone growth and increasing bone density.

Description

ACTIVIN-ACTRIIA ANTAGONISTS AND USES FOR PROMOTING BONE

GROWTH

RELATED APPLICATIONS

The present application is a divisional application of Australian Application No. 2012238197, which is incorporated in its entirety herein by reference.

This application claims the benefit of U.S. Provisional Application Nos. 60/739,462, filed November 23, 2005, 60/783,322, filed March 17, 2006, and 60/844,855, filed September 15, 2006, which applications are hereby incorporated by referenced in their entireties.

BACKGROUND OF THE INVENTION

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Disorders of the bone, ranging from osteoporosis to fractures, represent a set of pathological states for which there are few effective pharmaceutical agents. Treatment instead focuses on physical and behavioral interventions, including immobilization, exercise and changes in diet. It would be beneficial to have therapeutic agents that promote bone growth and increase bone density for the purpose of treating a variety of bone disorders.

Bone growth and mineralization are dependent on the activities of two cell types, osteoclasts and osteoblasts, although chondrocytes and cells of the vasculature also participate in critical aspects of these processes. Developmentally, bone formation occurs through two mechanisms, endochondral ossification and intramembranous ossification, with the former responsible for longitudinal bone formation and the later responsible for the formation of topologically flat bones, such as the bones of the skull. Endochondral ossification requires the sequential formation and degradation of cartilaginous structures in the growth plates that serve as templates for the formation of osteoblasts, osteoclasts, the vasculature and subsequent mineralization. During intramembranous ossification, bone is formed directly in the connective tissues. Both processes require the infiltration of osteoblasts and subsequent matrix deposition.

Fractures and other structural disruptions of bone are healed through a process that, at least superficially, resembles the sequence of developmental events of osteogenesis, including the formation of cartilaginous tissue and subsequent .adaei&Usation, The process of fracture healing can occur in iweways.Dhvef:^ primary bone healing occurs without callus formation. Indirect or secondary hone healing occurs with a callus precursor step, Prii»iapr2?'lieal &e: refermation of mechanical eomlniniy across a closely-set disruption. tinder suitable conditions,: bone-fosorbing cells surrounding the disruption show & tunnelling msorpdve.response and esmblifo pathways for the penetration of blood; vessels and subsequent healing. Secondary healing of bones follows a process of hfilaummifon, sod callus formation, callusmiBerafisation and callus remodelling, it foe Inflammation stage, kaenmtuma and haeniordtage formatiou results from the disruption of periosteal: and endosteal blood vessels at foe site of upsry, InSammatory cells invade: the area, fe soft callus fopnaffou stage, foe cells produce aew vessels, SbrobiastSj latracellnlar maieriai and supporting eels, forming granulation tissue m &e spasc between:foe ftacture'ftn^nersts. Clinical union; across foe disruption is established by fibrous or cartilaginous tissue (soft callus}. Osteoblasts are formed and mediate the mir^l|^c«;o?S9fociSlk^wM«|t Is then replaced by lamellar boss ami subjected to the normal remodeling processes.

In addition to fractures and other physical disruptions of hobo struetbre, loss of hone mineral content and bone mass eon be caused by a wide variety of sondlfionnnnd may result in significant medical problems. Changes to bone mass occur in a relatively predictable way over foe life of an individual, tip to .shout age 30* bones of both men and women grow to maximal-mass through linear growth of foe endochondral growth plates and radial growth. After about age 30 (for trabecular bone, e.g,, fiat bones such as foe vertebrae and pelvis) and age 40 (for cortical bone* e,go long houesifouad in the limbs};, .slow hone loss occurs: la both men and women. In women* a final phase of substantial bone loss also occurs, probably due to ; postmenopausal estrogen defiolencies. During this phase, women. may lose an additional lO^-ofbtaspptitss-'-fiw foe cortical hone and 25% from fo© trabecular; compartment, Whether progressive bone loss results 1st a pathological eortdiiion such as osteoporosis depends largely on the Initial bone mass of tbs htdividtusi and whefoer there are exacerbating conditions.

Bone loss is sometimes· characterise# as an imbalance in foe normal bone remodeling process. Healthy hone m constantly sobjecf'fo remodeling, Remodeling begins with resorption of bone by osteoclasts. The resorbed bone is then replaced by new bone tissue, which is characterized by collagen formation by osteoblasts, and subsequent calcification. In healthy individuals the rates of resorption and formation are balanced. Osteoporosis is a chronic, progressive condition, marked by a shift towards resorption, resulting in an overall decrease in bone mass and bone mineralization. Osteoporosis in humans is preceded by clinical osteopenia (bone mineral density that is greater than one standard deviation but less than 2.5 standard deviations below the mean value for young adult bone). Worldwide, approximately 75 million people are at risk for osteoporosis.

Thus, methods for controlling the balance between osteoclast and osteoblast activity can be useful for promoting the healing of fractures and other damage to bone as well as the treatment of disorders, such as osteoporosis, associated with loss of bone mass and bone mineralization.

With respect to osteoporosis, estrogen, calcitonin, osteocalcin with vitamin K, or high doses of dietary calcium are all used as therapeutic interventions. Other therapeutic approaches to osteoporosis include bisphosphonates, parathyroid hormone, calcimimetics, statins, anabolic steroids, lanthanum and strontium salts, and sodium fluoride. Such therapeutics, however, are often associated with undesirable side effects.

In one embodiment, the present invention provides compositions and methods for promoting bone growth and mineralization.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

SUMMARY OF THE INVENTION

According to a first aspect, the present invention provides a polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 3, wherein the N-terminus of the polypeptide is ILGRSETQE.

According to a second aspect, the present invention provides a pharmaceutical preparation comprising the polypeptide of the first aspect and at least one pharmaceutically acceptable carrier

According to a third aspect, the present invention provides an expression vector comprising a polynucleotide comprising a coding sequence for the polypeptide of the first aspect.

According to a fourth aspect, the present invention provides a cell comprising the vector of the third aspect.

According to a fifth aspect, the present invention provides a method for promoting bone growth, increasing bone density, increasing bone strength, treating or preventing a bone-related disorder, promoting bone growth and inhibiting bone resorption, inducing cartilage formation, increasing bone mineralization, preventing demineralization of bone, treating a bone fracture, or treating a cartilage defect in a subject, the method comprising administering to a patient in need thereof an effective amount of a polypeptide of the first aspect.

According to a sixth aspect, the present invention provides a method of making an ActRIIa polypeptide, comprising: a) culturing a cell under conditions suitable for expression of the ActRIIa polypeptide, wherein the cell comprises vector of the third aspect; b) recovering the ActRIIa polypeptide so expressed.

According to a seventh aspect, the present invention provides an ActRIIa polypeptide when produced by the method of the sixth aspect.

According to an eighth aspect, the present invention provides use of a polypeptide of the first aspect for the manufacture of a medicament for promoting bone growth, increasing bone density, increasing bone strength, treating or preventing a bone-related disorder, promoting bone growth and inhibiting bone resorption, inducing cartilage formation, increasing bone mineralization, preventing demineralization of bone, treating a bone fracture, or treating a cartilage defect.

Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

In part, the disclosure demonstrates that molecules having activin or ActRIIa antagonist activity (“activin antagonists” and “ActRIIa antagonists”) can be used to increase bone density, promote bone growth, and/or increase bone strength. In particular, the disclosure demonstrates that a soluble form of ActRIIa acts as an inhibitor of activin-ActRIIa signalling and promotes increased bone density, bone growth, and bone strength in vivo. While most pharmaceutical agents that promote bone growth or inhibit bone loss act as either anti-catabolic agents (also commonly referred to as “catabolic agents”) (e.g., bisphosphonates) or anabolic agents (e.g. parafeyroid hessaoae, F1B> when appropriately dosed), the soluble ActRJIa protein exhibits das] activity, having both eatsbolle and anabolic effects. Thus, the disclosure establishes that antagonists of the activin-AntKjla signaling pathway may he used to ««ease hone density and promote hone growth. While soluble ActRlfa may affect bone through a mechanism other than activin antagonism, the disclosure nonetheless demonstrates that desirable therapeutic agents may be selected oa the basis of art aetivin~.4cifellh sttiagonisi activity, therefore, In certain embodiments, the disclosure provides methods tor using: actsvIo-AotlSla antagonists, including, .tor. example, aeilvindhnding AelBIfa polypeptides, anfhaeirem antibodies, antl-ActfeOa antibodies,: activin- or: ActMIla^tatgetbd small moieehles and aptamers, and uudeic addhfhM decrease expreasioh ofoetlymind. with low bone density or lOW bone strength, such as osteoporosis, or to promote hone growth in patients m need fheteofi: such as in patients having ihone hacture:,: Additionally, the soluble AetSJIa polypeptide 'pfomofes hone growth without causing a: eotisistentiy measurable· Increase in. muscle mass in certabt aspects, the disclosure provides polypeptides ccanprising a soluble, aotivm-bindmg ActRlla polypeptide feat binds to activin. ActRlfa polypeptides may be formulated as a plmtmaeentieai preparation comprising the actMatemding AeiKila polypeptide and a pharmaceutically acceptable carrier, Ihpfetably, the aeiiyinrhhidmg ActRlla polypeptide binds to .activin .-with a 1½ less than 1 nnemmolar or less than 100,10 or 1 aanonmter. Optionally, the activitwhindlng ActMIa polypeptide selectively binds activin versos OOP! 1 and/or GX>FS, and preferably with a Kb that is at least Itl-fbld, dbdbidpr Sd-fold lower with respect to activin than with respect to GDF1.1 and/or tSDFS, White not wishmg to be bound to a particular mechanism .of action, it is expected that tills degree of selectivity for activin inhibition over GBFi'l/ODFS inhibition accounts for the selective effect eh hone without a consistently measurable effect on muscle. in many embodiments, an ActMIa polypeptide will be selected for causing less than 15%,Jess than 10% or less than S% increase In muscle at doses that achieve desirable effects pn bone. Preferably the composition is at least 95%purei, with respect to other poly pepti de components, as assessed by size exclusion chromatography, and more preferably, the composition is at ieasfR8% pnre. An activimbindlng AetRJIa polypeptide for use in such--a preparation may fee- those sack as a p@l)f«ptide:h&amp;#ig'an;aa^^;acid sequence selected; fiem SBQIX> NQs; 2,3,7 or 12, or having an amino seM seqnerere that Is at least S0%, M%» 98%·» 95%» 97% or 99% identical to an amkm: acid sequence selected front SBQ ID NOs; 2, 3> "7,12 or 13. An actlvin^indipg ActRiia polypeptide may include afoneiionaiimgmemofa natural ActRlia polypeptide, such as one comprising at least 10,20' or 30 amino acids ofa sequenceselected from SEQ ID NOs: 1-3 or a sequence of SEQ ID NO; 2, laekingthe CAerminal 10 to 15 amino aelds (tlmtkaif*). A soluble, aetivia-bihdrag AotRila polypeptide may indn<le one dr more alterations In Ike ammo acid sequence (e»g„ in the liland-dsmding domam| mlative to a naturally dfeutring: AetRIIa polypeptide/ Examples of altered AetRIIa WO 2006/012627, pp. 59-6¾ meorporeied by reference herein. The aitereboa in the amino acid sequence may. Tor example, alter giyeosylatlon of the polypeptide when prodnced in a mammalian, insect or other cleavage of the polypeptide relative to die naturally occurring AelRIIa polypeptide.

An aetlvinmmdihg ActEifa poiypeptide nray be a fusion protein that has, as one domain, an AeiRIla polypeptide (e>g,s a Mgpd-biodiOg portion of an Ao®Ha) and one or more additional domains mat provide: a: desirable property,: snob as improved phararacokmetics, easier purMdi^pji^::p^l«| Id: particular tissues*. etc, For example, a domain of a· fusion protein may enhance one Of more of in vivo stability,; mi vivo half life, aptafcc/admiaistraiion,. tissue localkaiion or disPibution, formation: of protein complexes, moitlmerkatidn df the fusion protein, and/or purifieabon, An aetivinfemdiug AetRIIa fusion protein may include an immuuogiohulm lc domain (wild-type or mutant) or a serum albumin or other polypeptide portion that provides desirable properties snobas improved

Ton preferred positioned between the f e ^sep^.;ae4:;i^:.extrsc«Etdar. ActRlia domain. T his nnstrectured linker may correspond to the roughly IS amino acid tmstruemred region at the Cdetminui end of the extracellular domain of ActRlia (the “tail”), or it Stay be an artificial sequence of 1, 2, 3» 4 or 5 amino :acids of alengih ofbetween 5 and 1-1,.¾¾ 30, 50 or mors amino aaicis tltai are relatively .fee of secondary siructonii, pr a mixture of bo®. A linker may be rich in glycine asd. proline residues and may, for example, contain a single seqaeocs of fesousBe/senne and glycines or repeating sequences of feequmerseripe and glycines (e.g., TG^.or SG* singlets or repeats!, A fusion protein may include a purification subsequence, such as an epitope tag, a FLAG tag, a ppiyMstidhte sequence, and a GST fusion. Optionally a soluble ActRIIa polypeptide Includes one or more modified amino acid residues selected from: a glycosylated amino acid, a FEGylated amino acid, a furnesylated amino acid, an aeetyiatsd amino acid, a hioilnyiated amino acid, an amino acid conjugated to a lipid moiety, and an amino acid conjugated io an organic derivatfeing agent A pharmmsetdicaiprepaMionmay also amlude mm or more additional compounds such as a compound feat is used to treat a bmm disorder. Preferably, a pharmaecniical preparation is snbstanPally pyrogen fern in general, it is preferable: that an Aetidla protein be expressed in a mammalian ceil line feat mediates: suitably natural gfyeosyiabon of the AetSila protein so as to diminish the likelihood of an ntrfkv®able immune response in a patient Human and CHO eel! lines have been used suecefofoily, and it is expected that other common pmhmalian expression systems will he useful.

As designated ActROa-Fc (a form "with a minimal linker between the Antfolfa portion and the Fc portion) have desirable properties, including selective binding: to aetivin versus GDF8 and/or GGFIl, high affinity ligand binding and sermn Mif life greater than two wedcs in animal models. In certain provides ActEHa-Pc polypeptides and pharmaceutical preparations comprising such polypeptides arid a pharmaceutically acceptable iCxcipient.

In certain aspects, the disclosure provides nucleic acids encoding a soluble: actlvimbinding ActRila polypeptide. An isolated polynucleotide may comprise a coding sequence for a soluble^ actiyinfeindiug ActRIIa polypeptide, such as described;-above. For example, m isolated nucleic acid may include a sequence coding for an exteaeeilnkr domain (e ,g., ligand-binding domain) of an ActRIIa and a sequence that woul d code for pari or all of fee traasmembrane domain and/or the cytoplasmic domain of an AetRSaybm for a stop codon positioned within fee riansmembmne donminor theeytopiasipic domain, or positioned between the iransmembrarse domain or cytoplasmic domain. For example, an isolated polynucleotide may comprise a fetlriengfo. AclRBa ppiynuelefade sequence such as SEQ IB M); 4 or 5, or a partially irutmated version, saidnsoluted polymMeOiafc fiather comprising a transcripthmiemnnation codon at. least six hundred nucleotides before the 3Meraxiuus or otherwise posHfoned such that translation ofthe polynucleotide gives pisefoan extracellular domain optionally fused to a irurmated pOttioH of a foiReugth ActRIla. A preferred nneleic acid sequence is SEQ ID N0; 14, 'Nucleic acids disclosed herein .may be operahiy linked to a promoter for «xpressimts add the diseiosnre pmvfdePeells transformed with such recombinant falyimeleotides. tfaforably the cell is a maofaalhm cell such as a CUB pelf. da certain aspects,, the disclosure provides methods for making; a:soluble, actiyiil-faiadmg ActRIla polypeptide, Such, a method may include expressing airy of the nucleic acids fag,} SEQ IB NOt 4,5 or 14} disciosed .hemm iu a sudabk cell such as a; Chinese Itamster ovary (€HG| cell. Such a, method may comprise: a) e uttering a: cell under conditions suitable for expression of die soluble Actftfla polypeptide, wherein said cell is tensformed with a soluble AetEIfa expression construct; mKi b) recovering the soluble AetRlIa polypeptide so expressed. Soluble Acthdfa polypeptides may he recovered as crude, paitlally purified or highly purified factions, fariftoaiion may be achieved by a series ofpuriffcafion steps, including- for example, one, two or d>ree or mom of the following, in any order; protein A chromatography, anion exchange chromatography fag., 0 ssphamse), hydroptobtc interaction ehrofoato^aphy fag., phenylsepharose), s|?e exe&amp;sion cfo'omatopaphy,: and cation, exchange eliromaiography, hr certain aspects, an aetivImAetMia antagonist: disclosed .hemin, such as a soluble, actiyIfobinding ActRIla polypeptide, may be used in a method for promo fag bonegmwth or increasing bone density In a subject ineertaia embodiments, foe disclosure provides methods for treating a dlsmder .associated with, low bone density, or to promote bone growth, in patients in need thereof A method may comprise administering m a eul^eet iir need : thereof an efafaye amount of adtivin-AefRfia antagbnisi. fa certain aspects, the disclosure provides ases of sMtvirwAeiEIIa amagUnist for making a msdioahteni far the treatment of a disorder or condition as desorihed harem. % certain aspects, the disclosure provides a method for identifying an agent that stimulates growth oil or increased mmeralinartsn oil bone. The method comprisesi a) identifying a test agent that: binds id activln or a Hgand-Mhdlhg dom ain of-an AetMih polypeptide; and h) evaluating the eMsei of the agent on growth άβ or inlnerahzatidvi of bone.

BRIEF pESCRIPTipM OF THE DRAWINGS

Figure 1. shows the purification of AclRIIa-hFc expresaed in CHO cells, The protein purifies as a single, web-defined peak.

Figure 2 shows the binding of ActRIIa-hFc to aetivin and G0F-41 s as measured by BiaGshd^ assay..

Figure 3 shows a schem atie for lire A-204 Reporter Gene Assajo The figure shows the Reporter vector: pGL3(CAGA)l2 (described in 'Deimler et al, 1998, EMBQJ.7: 3991-3100.) The CAGA12 nrntlfiis pmsmdin TGF-Beta responsive genes (PAI-1 gene), se this yeetor i s of general use for factors signaling through Smad 2 and 3. f igure 4 shows the eSects,:df AetRIia-hFc (diamonds) and: AcfiflIa“mFc |(sgpare8) :on.GGF“8 signaling in the: A-204 ileporter Gepe Assay, Befit proteins exhibited stihstanfal ffihftition of Gi3f~B mediated signaling at pieomolar concentrations.

Figure 5 shows tfee- :ι^ίΡ'ΐίϊ]κ^:'4^0Γ<Ι^Θ!ί’ on : GDf ~11 signaling In the Α-2Θ4 Reporter Geo© Assay.

Figure 6 shows examples of BERA images of control- and AetRIIa-mFc-treated BALB/c mice, before (top panels): and after (bottom panels) the 12-week treatment period. Paler shading Indicates: increased bone density* ,Ψ^^<ψ'φ^$Ρ·ίβ2Μ^.ύ(Μ of tfep effects of AetRIla-mFc on bone mineral density in BALB/c mice over the 1.2-week period. Treatments were control (diamonds),, 2 mg/kg dosing of ActROa-mFc (squares), 6 mg/kg dosing of ActRIla-mFo piangtes) and 1ft mg/kg dosing of AotRIia«Fc {cireles}.

Figure 8 shows a quantification of the effects of ActRila-mFc on bone mineral content In BALB/e mice over the 12-week period. Treatments worn control (dinmonds)j 2 mg^ dos|ng of AotRifenFe (squares), 6; mg/kg dosing of ActMia-mFc (triangles) and Iftmg/kg ActRl&amp;^nFc. (bimiss>).

Figure 9 shows a quantification of the eftpots of ActBIJa^mFc on bone mineral density of the trabecular bone inovarieeionfized (OY?0; .qfj^tpd (SHAM) €S7BIAniie« over after a (i-wsek period. . or 10 mg/kg dosing of AciSIIa-mFc (Aeplla).

Figure. 10 shows a quantification of the: effects of AciRilmmfc on the trabecular bone in ovariectomi&amp;sd (0V3C) FSFBLb mice oyer a 12-week period, Treatments were control (PSi; pale bats); or HI mg/kg dosing Of ActBlfa-mFc (AciBIla; dark bars).

Figure 11 shows a qoantiReaftoh of dm effects of ActRlla-mFo on the trabecular bone in sham operated C57.BL6 mice after 6 or 12 weeks o f treatmen t period . Treatments wem control (BBS; pale bars) or 10 mg/kg dosing of AetRfia-mFc (ActROa; dark bars).

Figure 12 shows the results of pQCT analysis of bone density in ovarieetomiaecl mice over 12 w<mks of treatment, Ifeahueftte word control (FBS;; pale Bars) or AcfRIImthFo (dark bars). y~axis: mg/eem

Figure 13 depicts ftro results of pQCT analysis of bone density in sham Operated mice over 12 weeks of treatment Tteatmeuts ware eoutrol (FBS; pale bars) or ActRlik-mFo fdark bars), y~apis; mg/eem:

Figures 14A.and 14B shnw>h6|e'hody12 weeks of treatment (A) and 0 y/vo analysis oFMaurs (B), Fight areas;: depict areas Of high, bone density

Figure 1.5 chows ex vivo pQOT analysis of the ismoral midshatt sfter twelve weeks of tresimeat. Treatments werevehicle control (FB:B>: dark bars) and AetRIla-mFu (pate: bars), Tbe four barsto the left show: total bone density while the fbur bam to rite right: show cortical bribe density . The first pair of bars in each set oftour bats represeat dais front ovarieetomixed mice while the second pair of hamraptetent data from sham operated mice,

Figum 16 shows aw vim pQCT analysis and diaphysea! hmie eomxait of the femoral nui^a&amp;'ajSPrfwe!# weeks of treatment, control (FBSj (pale bars). The total bone content while the tour bars to the right show conical bone content. The first pah of bars is each set of ibsr bars represent data the second pair of bars represent data bom sham operated mice.

Figure 17 shows ex vivo pt^CT analysis of thefemoral midshail and femoral oortieal thickness. Treatments were control |FBS» #tfe bars) and AoiKBamtFo (pale bars), The fear bass to fee fell show endosteal cimmnferenoe white the fenr bars to the right show periosteal cireumfemnee. The firstpair of bars in each set of fear bars repmsmd datefeom ovarfeetornizcd mice while tire second pair of bars represent data from sham operated mice.

Figure 18 depicts fee resutif of meoitenii^:fe^fog'-of femum-afel^vdlve weeks of treatmeut, Treatmente'We^iepnttttl^S^^disdfe'bgas) and AetRiiaHrnFc (pale bars). The Wvo'baiS'to:t^1bfei^present^^:^tn'0¥aTi&amp;Ptomi»KJi«iice ’while: the last two bars represent elate fetirn sham operated mice.

Figure: 19 sbows the effects of AetrllamtFe on trabecular boaoTolume,

Figure 20: shows the effects of AcirnnnhFc on trabecular architecture in fee distal femur.

Figure 21: shows the ebeots of Actrlla-mFc on eorbesl bone.

Figaro 22 showslho effects.of AcirH&amp;#Ff «#' the meehanteal strength Of bone.

Figure 23 shows the effects of different doses of .ActRffewhFc on bone characteristics at those different dosages.

Figure 2d shows bone histomorpbometry indicating that ActMia-mFo has dpal analolio and anti-resorptive aedyfty.

DETAILED DESCRIPTION' Of THE WENTIOH I. Overview

The transforming growth fecior-beta {TCIF-beta) superfomlly eQtttains a variety of growth Deters that sham cooimoo sequeoca elements and structural motifs. These j^tefns on a large variety of co!i:||^'%:|i<^':v^ehrat^' aod myetiehrates. M^hets- of perform important functions during embryonic development in pattern formation and tissue specification and can influence a variety of differenliafidn processes^ including: adipegeuests, myogenesls, choadrbgenesia, eardlogenesls, hematopoiesis,, neurogenesix, and epithelial cell differentiation, The family is divided into two genera! branches* the BMP/GDP and the TGF-beta/Aetivin/BMPIO branches, whose members have diverse, often complementary effects. By manipulating the activity of a member of the XGF-beta family* it is often possible to cause significant physiological changes in an organisim For example, the Piedmontese and Belgian Blue .cattle breeds cany a lossroBfintction mutation in the GDF8 (also called myostatin) gene that causes a marked inemase in mnsele mass. Qrobet et at., Nat Genet. 1997,17(1):71-4. Furthermore, in humans, inactive alleles of GDF8 are associated with increased; muscle mass and, reportedly, exeeptlonai strength, Sehuelke et ah, N Bngld Med 2(304,350:2682-8.

Aotivins are dimeric polypeptide growth factors that belong tothe TGP-beta superfsvmiy. There are three principle activin ferns (A, B, and AB) that, are homo/heterodimers of two closely related β subunits (felfe fePa> and |:3λΡη). The human genome also encodes an activin C and an activin E, which are primarily expressed in. the liver. In dre TOF-bete δίφΟΓΜηΙίρ, acfiyihs are unique and fe4tifimct|o«aI#ct^ that can sfimulate hornfeie production in o varian and placental cells, support neuronal cell survival,Jnfiuenee cell-cycle progress positively or negatively depending on cell type, and induce mesodermal diferentiation at least In amphibian enfeyds (DePaolo pt ah, 1991, Froe Soe Ep Biol Med, IbSDOO-fiafDyson et a!, 1997, ChrpBioL :7:81-84: Woodrdl 1998, Blochenv Pharmacol. 551953-963), fsgi&amp;r (BDP) isolated: from the stimulated human, monooytie leukemic cells was found, to be identical to aMy&amp;t A CMtipia et a I., 1988, FMAS, 85:2434). R has been suggested that aetMh A acts as arkfoml, positive fogidator of erythropoiesls in the hone marrow. In several tissues, activm signaling is akagooked by its related heterodimer, inhlbin. For example, during the release onblbcle-shrnukting hormone (FSH) ifom the ptfobary, aetivln promofcs FSB secretiim and synthesis, while Irkthin prevsnis FSB secretion and synthesis. Other proteins that may regulate to act!vihInclude foliistatsg {F§), foiiikatin- related protein (FSBP), ^-mdcrogiohidln. Cerberus, and endogiim TQF-β signals are mediated by heteromerle complexes of Wpel and type II serine/ threonine kinase receptors, which pbesphorykte and activate downstmam Smad proteins upon ligand stimulation (Massagne, 2000, Nat Rev, Mol, Cell Blob l:li9»T7S), These type I and type II 5wept0k:-ahe-^ksmembraiie'''p*btgtns> composed of a ligand-hlnding extracellular domain with cysteine-rich region, a transmembrane domain, and a cytoplasmic domain With predicted serine/threonlne speeiBcity. Type I receptors are essential for signaling; and type 11 receptors are required for binding ligands and for expression of type I receptors, Type I and II aetivin receptors fob» a stable complex after ligand binding, resetting in phosphorylation of type I receptors by type II receptors.

Two related type II receptors, ActRIia and ActRIIb, have been identified as thetype fl recept^rsTbr aetlviss (Mathews and Yaie. 1991., Csli 65:973-982; Attisano etaL, 1992:, Cell 68:97-108). Besides aefi vins, ActPJIa and ActRIIb can biochemically inieraet with several ptherTCF-β fondly proteins,iaeindin.g BMP?, Nodal CdlFI, and <3DF11 Ofomashrkk 1995,1 Cell Bid. 130:217-526; Lee and McPherron, 2001, Prom NfatL Acad. Sci 98:9306-9311; Yeomk Whitman, 21)01, Mol Cell 7:949-9S7; Gh et ah, 2002, Oeues Dev. 16:2749-54), AJLK4 m the primary type I receptor activins, pariioukrly for aetivin A, and ALK-7 may serve as a receptor for aeiivins as well, particularly for aetlvln B:

As demonstrated herein, a soluble AetRIk polypeptide (sAetRlIa), Which shows substantial preference in binding to aotivinA as opposed to other TGF-beta family members, such as GDF8 orGDFI i. Is effoklve fo pfomoie bone growth and increase bone density in vivo. White not wishing to be bound to any paMcuIar meohanishb it Is: expected that tile effect ofsA^MviS'-^^pilmarliy by aii activin antagonist effect, given the very strong aetivhl binding: fpicomolar dissociation constant:) exhibited by the particuiar sAntRIIa construct used m these studies, Hegardjess of mechanism, it is apparent: from the; data presented. herein that: Actlrifemetivin antagonists db: increase bone density loinortnal: mice and in mousd models for osteoporosis. It should be nomd that bonels a dynamic. tissue, with, growth or shrinkage and increased or deoteased density depending on a balance '.of factors that produce-bone and sthnulaie mlhefelkarioh |grimariiy osteoblasts) and factors that destroy and demineralize bone (primarily osteoclasis). Bone growth and mineraiiaaiion maybe increased by mcrea§kg'^p,pp^^ -by decreasing: the destructive factors, or both. The terms ^promote bone .gtowthfi and increase hone .mineralisation” refer to the observable physical changes in bone and are intended: to: he neutral as to thcunechanism by which changes in bone occur.

The mouse models/for osteoporosis: and: bone growih/denslty th® were used in the studies described: herein are considered to be highly predictive of efficacy In humans, and: therefore, this disclosure provides methods for usmg AeiEIla polypeptides and other acrivin- ActRlIa aniagoniste to pidtnolse' increase boos density in hmnans, Acdyin-ActRIla actiyin^blnding soluble ActMta polypeptides, antibodies that hind to ncuivis (patticuiarly the aedvin A or B subunits, also referred to as βA or fiB) and disrupt ActRHa binding, antibodies that hind to Actiy&amp; and disrupt activin binding, non-antibody proteins selected fbr AgiBnalMndwjg (seee.g,,

WQ»2/0kSi7ls ^0^006/055619, :W<O/2OG2/032925, VS 2002/0133939, and tlS 2005/023k64# fc examples of such proteins and methods tor design and selecti<m of same), randnmined peptides selected for aefiyin or AeiRRa binding, often affcred to an ^ domain, Two difeeM poteins (or other moieties) with activin or AotRIXa binding activity, especially activin binders that. Mock the type I (e»g«* ^.solbblie ty^^au^s^-^eptor) and type it petty In receptor) binding sites, respeetiVeiy, may he Ihfed togetherto create a bifitnc&amp;nal binding mnleeule. Mueleic acid aptamers, small moiecales and other agents that Inhibit the ^vinTAc^Ik^^idl^axis. Various proteins have activin-' ActRIIa atUngonist activity, inckdihg inhihin fie., inhibin alpha subnnit), although ihhihift activin in all tissues, feliisiatm (e.g,, and fbliisraiinAtlSji,iferbems, FSRi> endbgiin, activin C, a!pha{2)~ maoroglobuiin, and an M108A (mefeonme to alanine change at gositimr 108) Matant aotlvin A> Gfemailyi alternative terms ofpairticulfely those with almfelonyMthe typetrefe^ hind to type Ilmcepmrsand tall fo form an active ternary eoniptex, thus acting as antagonists* Additionally, nucleic acids, such, as antisense molecules, slRN As or tibo^aesiltat Inhibit actlvitt A, B, Έ or E, or, particularly, ActlNfa expression, can be used as actiyln-AciEIIa antagonists. Preferably, the aetlvinAfetRila antagonist to be used will exhibit sdeotmty for inhibiting acfivlh-niediaied: signaling versus other' members; Of the TGE-heiallmiiy, and particularly with hsspeet to GDP&amp;and GDF! I. Soluble Actlllb proteins do bind tb aotlvin, however, the wild; type protein does not exhibit ' significant selectivity in binding to activin versus GDP8M R and preliminary experuhentS:suggest that this protein does not provide tire desired effects on bone, while also causing .substantial- muscle growifi, liowever, altered forma of AsffiBb with dilfeept binding properties have been identitied (see, e.g., WO 2006$ 12627, pp, 55 v59, Incorporated herein by refeenee) and these profess may achieve the desired effects on bone. Native or altered ActRllb may be given added specificity ipr activin % coupling with, a second, activin-seieefi ve binding agent

The terms used m this speclScatlon generally have their ordinary meaningshrthe art, within the context of this invention and in the ispeeltic context Where each term Is used, Certain terms are discussed below or elsewhere in the SpeelReMion, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and few to mafe and use them. The scope or meaning of any use of a term -will be: apparent fesm; the specific context in which the term is used. s*Ahour! and ' Approximately15 shall generally mean an acceptable degree of error for the .quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error ore within .20 percent (%), preferably within 10%, end; more preferably within 5% of a given value or range of values.

Alternatively, and padicuiariy in biological systems, the terms “about" aai “approximately” may mean values that are within -an order oilmagpit&amp;de, pfefombiy wMlua Srfcld apd more preferably within 2~&amp;ld «f a; givers value, Numerical quantities given fiereia are approximate unless stated otherwise, meaning that the term-“about” or “approximately" can be Infeed when not expressly stated.

The methods of the invention may· :aompasing sequences fo each other, including wild-type seqiemoe toone or umre; mutants (sequence variants), Such, comparisons typically comprise nlipmsents of polymer sequences;, e>g>, using: sequence alignment programs and/or algorithms; that are well-known# the art (far example, BLAST,: FASTA and MBOAIdGN, to; name a few), The skilled artisan can readily appreciate that, In such alignments, where: a mutation contains a residue msertlon or deletion the sequence: alignmeuf will introdncc a “gap” (typically represented hy p dash, or “A”) in the polymer sequence not containing the inserted or deleted: residue, forms and spel ling variations,, refers fe the relationship between two prmems that possess a “eommdh evolutionary origin,'5' including proteins -same species ofprganism, as well as homologous proteins from different, species of organism. Such proteins (and their encoding nucleic acids) have sequence limnology, as reflected hy their sequence similarity, whether In terms of percent identity or by tlmptesenee of specific residues or motifs and conserved positions. 2fe term “sequence sisniiafity,” in ail its grammatical forms, refers to the degree of identity dr eotrespohdence between nucleic· acid or amino acid sequences that may or may not share a common evolutionary origin.

However, in common usage and I» the instant applfeatlon, the term “homologous,” when modified with as adverb such as “highly," may refer to sequence similarity mid may ormay not relate to a common evokitionaiy origin. Z ActRlIa Polypeptides in certain aspects, the present invention relates to ActRila polypeptides. As used herein, the term “AetRIIa” refers to a family of aetivin receptor type lia (ActRila) proteins front any spades and variants derived from suchAetRIIa proteins by mutagenesis or other modification. Reference to AetRRa herein is understood to be a reference to any one: of fee eutteniiy Identified forms, Members of the AetRJIa family are generally Mnsinenferase preteinsj composed of a, iigandfoinding exteaeellulathem&amp;fo with a cysfelne-ridr region, a nnnsmernbrane domain, and a cytoplasmic domain with predieted serine /threonine kinase activity.

The term^lAotRIia polypeptide” includes polypeptides comprising any nataraliy oecumng: polypeptide of an Acfetllaifenily m ember as well as any varknts feefepf (incindlng nfetmttspi'agfeeniSj feSions., and peptidamlfeetic forms) feat retain a useful activity. For example, AetRRa polypeptides inetede polypeptides derived from fee sequence of any known ActRila having a sequence at least about §0% identical to the sequence of an ActRlIa polypepildRandpteferabiy at least 85% 90%, 95%, 97%, 99% or greater identity. For example, an Acfiffia polypeptide of the Invention may bind to and inhibit the fondiicm of an AetRlia protein and/or activity Preferably, an AeiRlfe polypeptide promotes bone grewfe and bone minetallKalion:, Examples precursor polypeptide (SEfe Π) ΜΘ: 1) and soluble human AetRlfe polypeptides (p.g,, SEQ ID Fifes: |,.3y? and 12),

The human AetRIla precursor protein sequence M as follows: oii^ics<ss WPl&amp;PPy YK1LLY3 L7PLMLI&amp;S l V l CAFWVYRHB KMAYEPVL VPTQD E A P PPPSP LEGLKFLOLX .S VKARGRFGC V WKAQ LLNSY VAVKTK' ? I QDRQSWQEE VEVYSLPGMKHENILQFIGAE KE.GTS VDV DLW LITAFilEKGS LS DFLKAteVVS¥9 NSLCH X ABTHAEGLA YLiiSOI FGLKPSilKPAISHRDI KSKNVLLKNNLTACIADFGLA LK foAGKSAGDTHGQV Of RRYMAP FYLE G&amp;IN FO n DA FLRI DM yAMG.'i.:V.LS9.HILASRCTA&amp;D.GP\f.DRYMLK; FBEBIGQ8 PS LS DMQE VWKKKKRPVLRDV^QKHAGMAMLCBTISSCWDIiDAEARLSAG CVGS RXTQMQRLTK1ITTSDfff7VTMVTMVDF?PKES3L (SBQ;ID::KO: l)

The signal peptide is single underlined; fee extracellular domain is in bold .and ihe potential N-linked giycosylation sites are double underlined.

The human AetRBa soluble (extracellular), processed polypeptide -sequence is as fellows:

I.IiGRSETQECLFFNAl'lWBKDRTNQTGVEPCYGOKDKERHCFAT WEH1SGSISIVKQGCWLDDTNCYDKTDCVEKEDSFEVYFCCCE gKMCBBRgSY FgBMBVTQPTStl E»VTPKPP <SEQ ID NO: 2)

The C-tenninai “taiP of fee extracellular domain is underlined. The sequence with the “tail” deleted (a Δ Ι5 sequence) is as fellows: ILGRSETQSCLFFHARWEKDETteQTGVEPCYGDKDKRRHCFAT WKGISGSIEIVKQGCWLDDIECYDSTDCVEKKDSPEVYFCCCE GHMCHEKFSYFPEM (SEQ ID NO:3)

The nucleic acid sequence encoding human AcfRITa precursor protein is as fellowsfeudeotides 164-1703 of Geubank entry NM_001616):

ATGGGA&amp;CTGCfGeAAAGXXGGCGXiXGCCSTCTXTCTXAXCTCOTGXXeFF CAGGXGCXAXACTTGGXAGATCAGASAGfCAGGAGTGTCTTTTCXXXAAXGC X AAX X GG G&amp;A&amp;A&amp;G&amp;CAG&amp;ACC&amp;ATCA&amp;AC TOG TGTTGAACGGXGX XAX G G X gag aaagat aaa cggcggcax tgtt x tgcxacc t cypiagaax axx xcx gg tg

CCAXxGAAATAGXGAAAGAAGaTTSTXGGCXGGATGATATCSACXGCXAXGA

CAGGACXGAXXGXGTAGAAAAAA&amp;AGACAGCCCXGAAGTATAXXXXXGXXGC

TGTGACiGGCtrUVXnTGTOTAaXGjaAXASTTTTCTTATTTTCCaGAG&amp;TGGXSG

TCFvCA.CAGCTX'ACTXCAR?teCCA.GXIACACCXteAG€CACCCXA7XAC:AAC'AT

CCAYATYXATTOerTGGTGCOACTXATGXTAATTGCGGGGATTGTCATXXGX

GCAXXXTGGGTGXACAGGCAXCACrteiGATGGCCTACCCTCCXGTACTTG'rTC

CAACTCAAGAGCCAGGACC&amp;CCCCCACeTTCTGCAXXACXAGGGXTGASACC

AGTGCXX7XAXXAGAAG7GA;WteAAGGGGAAGAXTTGGTTGTGTCTGGAAA

GCCeAGXTGCXXAACGAAXAXGXGGCXGTCAAAATAXXXCCAATAeAGGACA

AACAGTCATGGCAAAATG&amp;oTACGAAGTCTACAGXTTGCCTGGAATGA&amp;GCA

TGAGXmCATAXXXteAGXXC?teXGGXGCAGAAA&amp;ACGAGGCACCAGXG'rTG?^X

GTEGATCX??GGCTGSTGACAGCATTTCAXGAAA»GGGTTCA€TATCAGACI

XXCXXAAGGCTAA'i:GX<?.GXCTCXXGGXtecTGAACTGTGTGAXATTGCAGAAAC

CATGGCX.AGAGGAXXGGC&amp;XA'TTXACAXGAGGATAXACCTGGGCXAAAAGAX

GGCCACAAACCXGCCAXAXCXGACAGGGACATCA.AAAGXAAAAAXGXGCXGX

TG^GCilGGCAaGfCfQCriGGCG^TACCCkTGC^jCASCfr'TGGT^CCCGG^GG facafGSCTCCaS^GGTATTAGAGG^TGCTAT.^JVC'X'rCCMAGGGAXGCIVT TS'XTGAGG&amp;TAS&amp;TATGXATGCC&amp;TGGGAXT&amp;GTCCT&amp;TGGGAACTGGCTXC TeGCTGTACTGCTGCAGaTGGAvCXGTAGATGAATACATGTTGCC^'rTTGAG GACG3AAATGCA3CCAGD3TCCA!rC7;CTlA'iAAC:kCATGCAGGiAAGGTS'rL3'GTGC Sma^SMMGAGSCCTOTTTXMSAGATTATTGGCAGAAA.CA^GCtGGm* GGC.A&amp;eGCCCTGxGA&amp;ACCXTTG?ytGA&amp;TSG'XGGG&amp;ICACAACGCAGA&amp;CCG AGCl'T-AG CAGCXGG.&amp;T GX GxAGGXG AXAGASCf T&amp;CCCAG A T G C AG AG AC TX A C.tAXXTATTAT:rACte3GAXGAGCXCAT:rGGtA.ACGArG0XCX,0AATGGTG.ACXAA TGTXGACXXXCCtCCeAXXGXAGCX.ASfCTXIGA (SEQ ID NO: 4}

The nucleic· acid sequence encoding ulhuman AeiRIIa soluble (extracellular) polypeptide Is as.fellows; ATACT"CGTAGArCGGMAAGXGAGG;\GTGrG':'XTTCTGOAAGGCTAAXGGGG ••W.AAV.VG..\G'.:AA.CCAVr\.'AA-;·:i.·; GGGGTTGAAC λ..(.·\;ΧΌ T.AS'GG:':::A* :AG:;V-.G·: T&amp;&amp;&amp;C SGCG G CATGGC XT T GG T ACC T CA3 AAG AXT X T T XCTGGXT C GATT GAA ACsACXG.AG^ACAAGAiCTGXXGGCTGGSCl'tjATiVrCASCfGCGAXiGACijGGAC'iC ATGGTGXAGAAAAAAAAGACAGCCCTGAAGGATATXTTTGTTGCTGTGAGGG CAATAXGTGXAATSAAXAGTTTTCfTAXXTTWtAGAGAXGGXMTCXCACAG CGCACTXCAA&amp;TCCAGTTACACCTAAGCCACCC (SEQ ID NO: S) in a specific embodiment, the invention relates to soluble AeiRIIa polypeptides,. As described hereto, the term “soluble ActRIla polypeptide” generally refers to polypeptides comprising an extracellular domain of an AeiRIIa protein. The term “soluble Achilla polypeptide,” as used .herein, includes any naturally occurring extracellular domain, of an. AeiRIIa protein as well as any variants thereof (including mutants, fragments and pepiidomimeiie forms). An activin-bmding ActRHa polypeptide is one that retains the ability to bind to aetivbi, particularly activla AA, AB or BB. Preferably, an activin-binding ActRIla. polypeptide will bind to acfivin AA with a dissociation constant of 1 «Μ or less. Amino acid sequences of human AeiRIIa precursor protein. Is provided· below. The extracellular domain of an AeiRIIa protein binds to activin and Is generally soluble, and thus can be termed a soluble, aotivfe.fefedtog ActRIla polypeptide. Examples of soluble, sctivm-blndmg ActRHa polypeptides include the soluble polypeptide Illustrated la SEQ ID NOs:.2? 3,7,12 and 13. SEQ ID NO:? is pferred to as

AetEfitehEteand Is described fiirtherih the Bkamples, Qiher examples of soluble, ^i¥^bIbd3ag,ActRlIapoIypepi^s comprise h sigMl sequence in addition to the extracellular dote» «fan AoiRIIU protein* for example, the honey bee roellltin leader sequence {SEQID MO : 8), the tissue piammogen activator (TPA) leader ffiFQP NQh$) ar the native ActRIia leader (SEQ ID NO: 10). The AciRTIa-hFc polypeptide llhistrated in SEQ ID NQ:I3 uses a XPA leader.

Functionally active fragmetos of AetBfla polypeptides can be obtained by screening po!ypeptides: recombiaantiy produced from the oorresponding ifagineiit of the nucleic acid encoding an AptRIia polypeptide, in addition, fiagmentsean be chemically synthesized uringteefenkpes known in the: art such as conventional Merrificid solid phase ildoc or tffioe cheat istiy, Tbs teagments can be: produced^ (recombinastiy or by chemical synthesis) and tested to identity those peptldyl fagmente:^t^wil»'ae^0n\is's»tep®l8ls (tniiibhors) of AotBIla protein or signaling mediated fey acti via.

Functionally active variants of ActRUa polypeptides can be obtained by screening Bferaties of modified ^lyp^dtfes.t^omhfeantJl' produced from the correspanding n.rteagerfi5sed nucleic acids encoding an AetRifa polypeptide. The variants can be prOdneed and tested MtegoMste (inhibitors) of AetRIia protein or signaling mediated by activin. In certain embodiments, a functions! variant of the ActRIIa polypeptides comprises an amino acid 'seques^e-iiiat:Is at least 75% identical to an amino acid sequence selected from SEQ· ID MQs; Tor 3. In certain sases, the hmetiooal variant has an amino acid sequence afieast 8¾ 85%s, 9mM% 97%MK 9mm 100¾ identical to an amino acid sequence selected born SEQ TP NOsrTor $,

Functional variates may be generated % modifyn3g the sirijotere ofart AetRlte poiypeptideTbr Such purposes as enhancing thetepeutie efficacy, or stability (s.g., ex vivo shelf life and msistenoe to proteolytic degradation in vivo), Such modified AetRila polypeptides when selected to fetain activln binding, are eonstdmed: iteictianal equivaientsoi the natorali>f-oceurrmg AetEilafolypeiTfdete Modified AteRlla polypeptides can also fee produced, fer instance, by amino acid substitution, deletion, or addition. For instance, it Is reasonable to expect that an

Isolated replacement of a lenoin® Adth an IsOkueine or valine, an aspartate WttSt a .glutamate, a ihmoaloe with a serine, % δ *M$ w|$* a structurally related ammo acid (eg;,, porturtvative mtdaltousj will not have a sitdidr effect on the biological activity of the tosuiimg amkcuis. Conservative repkeemems am those that take .place within a lamify of amino acids that are related, m their sldeoh&amp;ins, Whether a change in the amino aeid:$ectuence of an ActRJia pplypepty® results in a fimefionai hQmotog eah.be reudfiy determined byassessing: :the ability of the variant ActRXIa ^.:|jrpdlu«es:.iBt is cells is: a feshtdn similar to the wild-type AcifiXia polypeptide, la certain embodiments, the present invention cositemplatas specific mutations of the ActRfla polypeptides so as to alter the giycosylation of the polypeptide. Such mthatfons may he selected so as to ratroduee or eUmisate one or more glycosvlatioo sites, such as O-linked orfehHnksd gfyeosyMon sites. Asparagine-linked glycosyktion recognition sites generally comprise a tripeptide sequence. asparagiafidX-threosine (or aspiragiaes-X-serihe} (where "A?5 is any ammo geld) which is specifically recognized fey appropriate cellular giyeesylafion enzymes. Idmaltemtion may also/foe made by the addition of, or substitution by,. ms or more serine Of tfeosme residues to tire sequence of the wild-type ActRllk polypeptide (for Odihted glycosyladen sites) - A ^jfety of amiso acid substitutions or dele&amp;pa at aoe or both of the ftmt or third amino acid positions of a glycdsylatioo recognition site {and/or amino acid deletion at the secOitd position) results in non-giycosylatkai at the modified tripeptide sequence. Another means of increasing the number of earhohydrate moieties on an AcfROa polypeptide is fey chemical or enzymatic coupling of glyeosides to fhs AciRila polypeptide, Depending on the coupling mode used, the shgar(s} may he attached to (a) arglmhe: and histidine; (h) fee carboxyl groups; (c) free snifhydfy!. groups such as those of cysteine; (d) free hydroxyl gtonps such as those of serine, threonine, or hydroxypmfine' (e) aromatic residues such as those of phenylalanine, tyrosine, or tryjAnphao; or (t) the amide group of glutamine, lihese methods am described in WO 87/05330 published Sep, 11,1987, and In ApBn and WrMon (1981} CR.C Crit. Rev. Bifedbem., pp, 2S9-30fe5 ineordofated hy refeence herein, Removal of ρηρ or more earhohydrate moieties present on an ActRIfa pofj'peptide may bo accomplished chemically a&amp;d/or emgmiatfeMly, Chemical degiyecsyistida may involve, for example, exposure of the ActRIIs polypeptide to the compound infioomfpeihaoesuifonie acid, or an equivalent coMpoimd. Sis treatment results in the cleavage of most or all sugars except the linking supr it^aeetyiglneosamlne or N-aeotylgsiaetosamihe), while leaving the amino acid sequence intact Chemical hegiycesylatian istimher described by lM:imuddihM: ai> 0987) Arch, Bioehem, Biophys. 2S9:S2andhyldgeetal (1981) Anal. Bioeltem, 118:131. BnaymaBe cleavage of carbohydrate moieties on AotBIla polypeptides can he achieved by the use of a variety of eudo- and exo-giyeosidases as desorihed hy Thotaknra et ai. (1987) MMnEnxymoL 138:350. The sequence of an ActRlia polypepPde may be adjusted, as appropriate, depending on the type <TexpMSsIon #stehi used, as mammalian, yeast, insect and plant ceils may all imrodnce differing giycosyiation patterns that can he atfoeted by the amino aci d sequence of the peptide, In general, Ao®lTa proteins tor use in hamass Mil be expressed in a mammahan cell line that provides proper glycosyiation, such as HEK293 or C1-1S) cell lines, although other mammalian expression cell lines, yeast cell lines with engineered glycosyiation enxymes and insect ceils are expected, to be useful as well,

This disclosure: further contemplates a method of generating mutants, particularly sets of combinatorial mutants of an AciBlIa. polypeptide, as weil as truncation; mutants; pools of combinatorial mutants are especially useful for identifying functional variant: sequences, lire purpose of screening such combinatorial libraries may he to generate, for example, AstMla polypeptide variants which, can act as either agonists or antagonist, or alternatively, which possess novel activities all together, A variety of screening assays are provided below, and such assays may be used: to;evaluate variants. For example, an AetSBa polypepfido variant: may be screened for ability to bind, to m AcfMla ligand, fo prevent binding of an AetlUla ligand to an ActRIIa polypeptide or to interfere with signaling caused by an Aet&amp;IIa ligand.

Ihe activity of an ActRlla palypeptide or Its variants may also be tested in a Cell-based or in vivo assay. For example, the effect of an ActElfo polypeptide variant on tlie expression: Of genes involved in hone prodnetion or bone destruction:: may be assessed. This may, as needed, be performed In the presence of one Or more mbomhlnam ActSIla ligand proteins (e.g., sethdn), and cells ma| Be transfected so as to produce ati AefRHa polypeptide and/or vanants ikereoi^ opdcnally, an AetRIla ligand, likewise, ah ActRIIa polypeptide may fee administered to a mouse oroide? ammalyand one or more Bone properties, stick as density1 or volume may be assessed, 1¾¾ Pealing rate for bone fractares may also be evaluated. Daal-eaergy x-ray absorptiometry (DEXA) is a well-established^ non-invasive, quantitative technique B>r assessing bone density in an animal, in immans central DEXA systems may be used to evaluate bone density in the spine and pelvis, These are the best pmdietors of overall bene density. Eeripheral DEXA systems may be used to evaluate bone density in peripheral hones, including, hand, wrtst, ankle and loot traditional x-ray imaging systemsi mcinding CAT scans, may be used to evalnate bone growth and fracture healing, Themeelameal strength of hone may also be evaluated,

Combinatorially-derived variants can be generated which have a selective or generally increased potency relative to a nafetally occurring AciRITa polypeptide. Likewise, mutagenesis can give rise to variants which have intracellular halMives dramatically different than the corresponding a wild-type AetRdia polypeptide. For example, the altered protein can fee rendered either more stable dr less stable to proteolytic degradation: Or other cellular processes which result nt desh uction of, or otherwise ioamlysdon of a native Ac iMIa polypeptide. Snob variants, and the genes which encode them;, can Be utilized to gjfer A^^a-pplype||fed0'-i^ls;fey modulating: the iaif-life of the AetRlla polypeptides. For Instance, a short half-life can give rise to more transient biological; effects and can allow titter control of recombinant AetRlla polypeptide levels within fee patient $s an Po fusion pnotern, inutationsmay be made in the linker (if any) and/or the Eo portion to alter the half-life of the protein, , A combinatorial library may be produced By way of a degenerate library of genes encoding a library of polypeptides which each include at least a pardon of potential A ctllila polypeptide sequences. For instance, a mixture of synthetic oligonucleotides cats be enzymatically ligated into: gene sequences such that the degenerate set of potential AclEila polypepMe hneieotide sequences am expressible as : individual polypeptides, or alternatively, as a set of larger fusion pfotems (e.g,, far phage display).

There are naany ways by which the library of potential homologs cm be genarateddrom a degeceraie bligoaeeleohde sequence. Cliemicatsyirthesls of a degenerate gene sequence can be carried out do amantoioatlc DBA syniheaij£er:i and (M: synthetic genes :ttpn be ligated Into an. approimiaic vector for exitressian, The sybthesis o|degejieiate oligonucleotides Is well known in the arh(see for example, Narang; SA ¢1.9^3} Tetrahedrop 39:3; ItakufaetaL, (1981) Recombinant DMA, Proe, 3rd Cleveland S^®#dSvhfecromebcuIess eb- AG Walton, Ainstetbamt Elssfler ρ§$73«2$9;;$^ιβ&amp;'ei aL (1984) Anno. Rev Hambem, 53:323;;ltahbra.et ah, 0984} Science 198; 1:056; Ike etat, (1983) HpCleic Aeld.iReb ll:47?)< Snell techniques have beep employed in the protelns (see:,ibr exmnple, So0tt et ah, (1990) Science 249:386-390; Roberts et ah, (1992) FNAS USA 89:2429-2433; Devlin et al, (1990) Science 249: 404-400; Cwirlact ah, (1990) lmS USA; 87; 6378-6382; as «las ITS. Paientldos; 3,223,409, 5,198,346, and 3,096(815).

Alternatively: ether fb»»S: o(mu^gesesir<»s,l«?.':»tii.i2»d to generate a combinatorial library. For example, AmRlIa polypeptide variants can he generated and isolated irotn a library by screening using, ibr example, alanine scmnritig mutagenesis and :ihe like (Rafei ai., (1994)Bicelmndstw 33:! 563-1572p Wang et ah, (1994) T Blah Chem. 269:3095-3099; Balini et ai, (1993) Gene 137:109-118; Grodberg et ah, (1993) Bar, ,1, Biochem, 218:597-601; Bsgashinm et ah, (1993) J, Biol, Orem, 268:2888-2892;: Imwmanet ah, (1991) Bmdiemlsby 30:10832-10838; and CtEnninghain et ah, (1989) Science 244:1081-1085), by linker scann&amp;g mutagenesis (Oustin'et a!., {1993) Virology 193:653-660; Brown et ah, (1992) MoL Ceil Biol. 12:2644-2652; MBEnlght et al., (1982) Science 232:316); by saturation mutagenesis (Meyers et at, (1986) Science 232:613); by BCR mutagenesis (hcimg et al,, (1989) Method Cell Mol Bio! 1:11-19): or by random, mumgenesls, Inchiding chemical mutagenesis, ate. (Miller et ah, (1992) A. Sbort Cmjrse In Bacterial Genetics, CSBL tress, Cold Spring Harbor, BY; and Greener et at, (1994) Strategies,m Mol Biol 7:33-34), tinker scanning iniaa^nesis, paBicularly In a combinatorial setting, is an attractive method tbr ideofilymg truncated (bioaetlve) forms of ActRUa polypeptides.

Awide tango of tee&amp;nkfues are known In the art for screening gene products of combinatorial iibmries made by point imttations and tarnations, and, &amp; that matter, for screening af>NA libraries for gene products having a certain property. Rtmh teehrnques will be generally adaptable fer rapid screening of the gene libraries; generated by the combinatorial mutagenesi s of ActRIla polypeptides, 'Ore most widely used"^l^u^:#r screening large gene libraries typioally «««pases cloning the gene Mbmry into replicable expresslopi'^iaiSjtt^iis^^feg appropriate cells with the resnlfingJlbrary of vectors, and expmssing the comknatonai genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encorhng the gene whose assays indnde amiym binding assays and aetivimamdlaAd cell signaling assays.

In certain embodiments, the ActRIla polypeptides of the invention may tojfher in addition to any that am naturally present: in the ActE|Ia polypeptides, Sdch modifications include, bar are not limited to, acdtylaiibn, earboxykiimf iglycbsyiadon, phosphoryladoh, iipldatlon, and acylation. As a result, the pplyji^dea may acid elements, such as polyethylene glycols, lipids,' poly- or mono-saoeharide, and phosphates. Effects of SoohlnOn^atnlno acid elements pnibe Ihnctionality of a ActRIla polypeptide tnay he tested ns described herein tor other AnfEfa polypeptide variants. When an ActRIla, polypeptide ^produced In cells by cleavinga nascent form. of the Ae®II&amp; polypeptide, post-traaslatlonal processing may also be important for correct folding and/or^ function of the protein, Differsdi. ceils (such as CHO, EeLa, MDCK, :293, W13S, ΝΙΚΑΙΑ orHBK2&amp;3} have specific cellular machinery and characteristic mechanisms tor si#rp0sHranstdtione.i activities and may be chosen to ensure the correct modiScatiomaBd: processing of fhe ActRIla polypeptides,:

In eeriain: aspects, functional variants or modified terms of the ActRIla polypeptides include fusion ptoteins having at least a portion of toe ActRIla : polypeptides and one or more fusion domains,. Well known: examples of such fusion: domains, include, but are sol limited to, polyhlshdine, Glu-GI a, glutathione S transferase (GST), iMoredoxlEs protein At protein G, m mimanoghfeuhE heavy chain constant region pc), maltose binding protein (MBP), or human seruEt albumin, A fusion domain may he selected so as to confer a desired property, For exaiEple, some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography. For fee purpose of affinity purification* reievaMmaMo^fbr affinity drromatography,· tdbh as: glutathione”,amylase-, and nickel- Or co balt” temp «gated resins are us&amp;dL Marty of such matrices are available In ^ti* forth, such as the Fharmaciu GST ptrlifeation system and the QiAeapress^M System |^lage«) useM with (HISs) fusion partners. As another example* a fission: domain Elay be selected so as to facilitate defection of the Aetllla polypeptides. Examples of such detection domains Include the various ilhoreseehf proteins (&amp;gk GPP) as well as'^eplto^ta^^which are usually short peptide se^feifees for which a. specific antibody is available, Well known epitope tags for which speclfls monoclonal antibodies me readily available includeFBAG, IPfluemst virus haeroaggl «tlhm (HA), «Ed e-myc tags. In some ea$esy the fesiort domains have a protease cleavage 'Fac^carl^;. or Thrombin, which allows the rcievant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins thereSom. d^''^^']be'.p)lbte-d.'^eint the fhrioh domain by Is certain preferred embodiments, an ActEllb polypeptide isfesed with a domaih that stablbKes the AetRIia polypeptide id vivo (a 'Mahlliber” domatu}. By “stahiiklpg55 is meant any thing that Increases serum half 1¾ fegardless of Whether this Is because of decreased destruction, decreased clearance by the Sddney, or ofhefphumiacokinetic effect. Fusions with the pc portion of ap ibiEumoglabnltu are known to eonler desirable pharmacokinetic properties on a wide range of proteins, likewise, fesrops to human serum albumin can confer desirable properties. Other types of fusion domains, that may fee selected Include mubimerfemg (e,g,s d jn^jsipg, domains and fiuietionai domains (that;confer an additional biological function, such as ferthor stlmnlatioR of bone growth or muscle growth, as desired),

As .a specific axaajpesiMpfeseriiffiMitioa provides a comprising a soiablei^g^iiute^Oimala P€ Aat^JIJaSsed if> an Fc domain ^&amp;g.» SBQ ID NO: 6). THTCPPCP&amp;FELLGG PSVFLFPPKPKDTLMX SRTPSVTCVVVD {A} VSEH.DFEVKF KWyVDQVHn/HNAKTKPeEBQFHSTYRWSXFTVEHQWL^SKSFKCKiE::):VSMK&amp;L: P¥PIEKTI:SK|k;KS:SFREPQVyTLFPSFE|SMTKM0F:SLTCBVEGFFfSDISFStE;SI GQPEHMYKFTFPV'LDSDGPFFLYSELTYDKSRWQQGISfVFSCSVMHEMHECAEHiT. qk.s:Es:ee:ese* '•Optioaailyptfee FO domain has one or mom mntafions at rislduaa SiicF as Asp-265, lysine 322, and Asn-434, In certain cases, lie nlniaai Fc domain having:; one or more of these mnialions (e.g., Asp4MS mtaaiimf} has reddeed ability of ^dln§'|^;^'F%Tec<^^riektive to asvlldtype Fc domain, in other eases, the mutant Fg domain having one or more of these; mutations :|e.g,, Asu-434 mufidion) has Increased ability of blading to the MHC class brelatedrFe>-rceepmr {FgRK): relative to a. wlldtypslh’^domam- ft is midamtodd ffiat different elements of the fesion proteins may he consistent with the desired fimmlonaiity. For example, m AefMla polypeptide may be placed C^ermlnai to a heterologous domain, or, afteroauveiy, a heterologous domain may be placed CAermmal to an AciRjia polypeptide- The AetBIfa polypeptide domain and the heterologous domain need not be adjacentin a fusion protein, and additional domains or ajnmo acid sequences may be inelnded C~ or hiAennmni to either domain Of between the domains.

In certain embodiments, the AcifiXla polypeptides of the present invention contain one or momModifleationsihat are capable of stabilizing the AetSIia polypeptides. For example, sueh modifications enhance the in vitro; half life of she ActMla poiypepfides, etdianee clroalatory half life of the AetRHa polypeptides or redtme pmteoiyiic depadanon of the: ActRIfa polypeptides. Such slabllizing; modifications Include, but are not limited to, fission proteins (mclndmg, ibr example,; fusion proteins comprising an AetRHa polypeptide and a stabilizer domain}, modifications of a giyoosyiation site (including, for example, addition of a gtycosyiaiton stfe-W'te ActRIIa polypeptide), and modifications oficarbfifeyfate moiety (inciuding^fot example, remo val of caybdiydmie moieties from an ActRIIa polypeptide), In the case of fusion proteins, an ActRIIa polypeptide Is fused to a riablileer domain sash as an IgG molecule (e.g., an Re domain}*:: As used Iieretiv fc term: “stabilizer domahfs not only refersto a fission domain {kg,* Fc) asfn the case of fitsion proteins,: hut also includes noj^fo^inac«3tt$'ib.mIito#^ such as a carbohydrate moiety* or nonproteinaceons polymer* snob as palpihylen©.'glycol.

In certain embodiments, the present invention makes available isolated and/br purified forms of fee ActRIIa polypeptides, which am Isolated from, or otherwise substantially doe of other proteins, AotRIIa polypeptides will generally be produced by expression from recombinant nucleic acids.

In eeitala aspects, tlrninvemton provides isolated and/or recombinant nucleic acids encoding any df file ActRIIa polypeptides (e.f., soluble ActRIIa polypeptides), including fragments, -fractional variants and fitsion proteins disclosed herein. For example, SEQ ID NO: 4 encodes the naturally oeeumag human ActRIIa precursor polypeptide, while SEQ ID MO: 5 encodes the processed extracellular domain of ActRIIa, The subject nucleic adds may be slngledtfanded or double stranded, Such nuclefe acids may be DMA or RMA molecules, These nndeic acids may be used, twf example, in methods for making ActRIIa polypeptides or as direct therapeutic agents (e,g,iln^a.gene-thempy'apptddch),:

In certain aspects, the::Subjesd--8m?fo|c' acids-^odmg Aslldlapdiypqptities lareferther understood to include nucleic .acids that are variants of SEQ ID MO: 4 or 5, Variant nueleoti.de sequences include;sequences that differ by one orrnore micfcotide: substitutions, additions or deletions, such as allelic variants.

In certain embodiments, the invention providesTsoIated or recombinant nucleic acid sequences that am at least 80%, 85%, 90%, 95%, 9?%* 98%, 99% Or 100% identical to SEQ ID MO: 4 or 5 , One of ordinary skill in : the art will apptecfefe fhat nncfeic aeid/sequences CompIemematy :to SEQTD MO: 4 or 5, and 'variants of SEQ ID NO: 4 or 5 are also within the scope of this Invention. In furtlier teribodteten%fite nucleic acid sequences of the invention can be isolated, recombinant, and/or fesed with a heterologous nucleotide sequence, or in «"0ΜΑ library. in other embodiments* mtetefe acids of the sequences feat hybridfee under highly stringent «>teditiohs %|^;ntideptiik|: sequence desxgnhied iu SEQ10'MO; 4 or 5, cemplemeM sequepc ef SEQ ID M0: 4 or 5, or hugneuis thereof M discussed ab#e,:qne of ordinary skill in the art will

which promote DNA hybridlaahen c^ he lped. O^ipfOrdmaty'Siili'in the art wlil understand readily that appropriate stringency conditions which promote DMA hyhrkiteation can be varied, For example, one could pta^mn the hyhddkatlon at 6.0 x sodium chlorlde/soditOT citrate at about 45 °C, tollowed by a wash of 2,0 x. SBC at 50 Far example, the salt eoheernmilon in the wash step can be selected from a low stringency of about 2l0 x SSC at 50 ¾ to a high stringency of about 0,2 x SSC at 50 "C, in addition, the temperature In the wash step cau he isereased teem low stringency conditions at room temperature, about 22 °C, to high stemgensy conditions at about 61¾. Both teinpersture and salt may be varied, or temperature or salt concentration may he held constant white the other varlabie is changed. In one embodiment, 'the: Invention provides nucleic acids which hybridise under low stringency conditions:: qf 6 X SSC at: roprh: temperaiure followed by a wash at 2 x SSC at room teOipetatuife

Isolated nucleic: acids which difter horn the nucleic acids as set rbrih in. SEQ ID NOs: 4 or 5 due to degeneracy in .the genetic code are also within the scope of fire invention.. For example,: &amp; number of amino acids are designated by more than one triplet. Codons that specify the Same amino acid, or synonyms (fe example, CA0 and CA.C ate: synonyms for Mstidiuefmuy result in ‘Ciionf “ mutations which: do not atSset : the amino acid sequence of the protein. However, it is expected that DMA sequence polyMorphlsms feat do lead to changes in the amino acid sequences of the sub|eet piwteins will exist among mammalian oeiis, Dne sMIIed hi the art will appreciate feat these variations in qifce.hhmoire':^ 3~5% ofthe nuoieotsdes) of the nucleic acids encoding a particular: protein may exist among individuals of a given species due to natural allelic variation, Any and all such nucdeotidfe variations andresuftfag amino acid polymorphisms are wiifcfn dm scope of tfjlsiirv'sntion, in eetialn eaib^ntenfe, the reecnahinant hnclek apids/pf the invention may bpoperbbfy linked to one on mam regulatory nucleotide sequences in nn: expression construct., Regulatory nucleotide sequences will generally be appropriate^ ceil need lor expression,: h&amp;merous appropriMe ^tsgslon vectors and suitable· repdatory sequences ate known1 In the art for a variety of dost cells. Typically»: said onp: Or more regulatory1 nueiootide sequences may mclnde9 but are not limited to, promoter sequences, leader or signal sequences, riboaomal binding sites, . ^arti.jandiit^rr^lnatlon.. translational start and termination sequences;, and enhancer or activator sequences, Constitutive or Iniducibte-:p0ta©^r8. as Imown Imtib art are eGOtempiatedby fee Invention, The jpnomoiejr§i;i»8i''-bG' eitte:«ai^liy •Gtkurring promoters, or hybrid promoters that combine elements eimomihanone promoter, .An expression constmet may be preseuf in a cell onun episome, such. as a plasmid, or the expmaslott construct may be inserted in a chromosome, lir a preferred embodiment. the cjqjression vector contains a selectable marker gene to allow the sefection oftransfbmied host cells. Selectable marker genes are well 1οκήνη;ΐη the art and Will vary with; the used.

In eerlaln aspects of the Invention, the expression vector comprising a nucleotide sequence encoding an ActRIla polypeptide and operably linked to at least one tegdatory sequence, Regulatory sequences are ar^recognized and are; selected to dinspf polypeptide. Accordingly, the tea» ^ulato^:$&amp;qu<^^::iilplnd^:|^not^ enhancers, and other expression eotdrol elements, Bxempiary regulatory sequences are described in Ooeddel; Germ Teehmhg$n M&amp;0md$ m Bn^mwlegy,

Acaderuk Press, San Biego, CA (lR9d)> Far instance, any of a wide variety of expression control sequences that control the expression of a IMA sequence When operatively linked to it may he used in these vectors to express BMA sequences eneudlng an AcfEIla polypeptide, Such useful expression control seciuenees. Include, for example, the early and late pfomotem of 8^4% M promoter, adenovirus or cytomegalovirus immediate early promoter, R$ V promoters,.the lae spteffijfoe trp system, the TAG' or TR.C system, T7 promoter whose expression Is directed by T? RNA polymerase, foe major operator and promoter regions ofphage lambda , the control regions for fi coat protein, tire promoter for S-phosphogiyeende kinase or other glycolytic es^mes, the promoters of acid phosphatase, e.g,, BhoS, foe promoters of the yeast amratiagi feors, the polyhedron promoter of foe haculoviras system sad other sequences known to controi the expression ofgenes of prokaryotic or etfoaryelic cells or their viruses, and various combinations fosreof it should he understood that the design of the -expression vetaor may depend on such factors as fee choice of the host cell to be bartsiormed and/or ike type of protein desired to be expressed. Moreover, the vectofs copy numbers the ability .t&amp;dda&amp;Oi· tetbcpy number and the: expression of any other protein encoded by the yeefofo such. as ahtibiotie markers, should also he considered.

A recoiisbinant nucleid:'^d:bf!ftier.«tv^tio»ibaft<he prbddeedtiy ligating the: cloned gene, or a portion thereof info: a vector suitable tor oppression, la either prokaryotic cells, eukaryotic cells (yeast, avian,: insect or mamtaalianl, or both. Expression vehicles for production of a recomhhiam AefRIfo poiypepfidc Include plasmids and other vectors. For instance, suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBl,-derived plasmids, pEX -der i ved plasm ids, p8Tac-derived plasmids and pIJC~deri ved plasmids fe expression in prokaryotic cells, such as E. colL

Some mammalian expression vectors contain both prokaryotie sequences to taellhaie foe prppgadon Of ^ vector m bacteria, and one or more eukaryotic transcription uulfo thid ere expressed In eufcarybtle cells, The peDNARamp, pefMAifoeo, pReAMVi pSV2gpt, pSY2neo, pSY2-dhfr, pTk2, pRSYueo, pMSC, pSV'f7, pko-neo and pHyg derived vectors am examples of mammalian expression vectors suitable for transfoction of cukaryotie cells. Some of these vectors are modified with sequences from bacterfol plasmids, such as pBR322, to facilitate replication and drug '0$ erfoatyotic cells.

Ahemativcly, derivatives of viruses such as foe bovine papilloma virus (BPY 4), or EpsteiifoBarr virus (pHBBq, pEEPtoerived fond ¢205} can be used for transient expression of proteins in. eukaryotic cells. Examples of other viral (including: systems can be jfeuad below fci tbo description ofgehfe therapy delivery isysfems. The various methods employed in the preparation of the plasmids and in fe&amp;hsformation of host organisms are well known m the art- For other suitable expression systems for both prokaryotic and euteiryotio «fells* as well as general recombinant procedures* See Molecuier ChrmigA lahorMor^ M&amp;mtat, 3rd E4y:ed, by Siimbtook, Priisch and M'aniatis pold Spring Harbor Laboratory Tress, 2061;),

In some Instances, it misy be desirable to express the recombinant polypeptides by the; rise of a: baeula virus expression system. Examples of such, baculovirus expression systems Include pVL-derived: vectors (such aS:p¥LI392s p¥L13$&amp; and p'WJdl:)9 pAeUW-derived vectors: |;su.cb as pAeUWl),» and pBIbeBae-dmved vectom (such as dreB-gai ooataihlngpBlneBaclH^

In a preferred embodiment» a; veetor wilfbe desigitedfer production of the street AotRlIa polypeptides mGHO ceils, such as a PcmvdSeript vector fStralagene, La Jolla» Calif,}» peDKAd vectors (Invitrogen, Carlsbad. Calif,} and pCI-neo vectors (Promega, Madison;, Wise.). As will be apparent» the subject gene cousdusts can be nsed to cause expression of the subject ActRIla polypeptides In cells propagated In endure, e,g>, to produce proteins, including fusion proteins or variant proteins» for purifieatkm.

This disclosure also pertains to a host cell transfected whh a recombinant gene including a coding sequence (e.g., SBQ ID NO: 4 or 5) for one or more of the snbjeet AetBHa polypeptides. The host celttnay lm any prokaryotic or eukaryotic cell, Eof example, an AetRIIApolypepiide of the invention may be expressed I» Meterml ceils (e.g* oslng a bacnlpvirns expression spteha% yeast, or manuSallah eells- Other suitable host cells am known to feose skilled in the art.

Accordingly, the present nwentlon further pertains to methods of producing the subject ActRiia polypeptides. For example, a host cell transfected with an expression vector encoding an ActRiia polypeptide can be cultured; tmder appropriate condftions to allow expression, of the. AetRHa polypeptide to occur. The ActRiia. polypeptide may be secreted and isolated feom a mixture of cells and medium cohtasnlng the ActRiia polypeptide» Alternatively, the ActRiia polypeptide may be retained. eytoplasmieafiy orfo a membrane ffoetiou and:the cells harvested, lysed. and the protein. isolated. &amp; cell culture includes Host cells, media and other Byproducts. Suitable inedla is- cell culture: are well known la thwart; The subject AcfRIIa polypeptides can be isolated fiom cell cuifore rnedfom, fioife ceils, or both, using techniques Idtewu la tire art for ptnifying proteins, laeludmg/ioit-sscbaugs chromatography,, gel fllirafidfi ehrmnatography, nhiafiltrafion5: electrophoresis, imnusnbaffinlty purification. with, antibodies specific for particular epitopes of the Actinia polypeptides and affinity purification with ao agent that binds to a domain fused to the AetRIla polypeptide (e.:go a protein A column may Be used to purify an Actfollafoe fusion); In a preferred embodiment, the Acfikila polypeptide is a fusion protein containing a domain which feeillfatcs its purification- In a preferred embodiment, purification is achieved by a series Of column chromatography Steps, including, for example, three or more of foe following;.: in any order: protein A chromatography, Q Sepharose chromatography; phsnylSephazose chromatography, size exclusion chromatography, and cation exchange chromatography . The purification could be completed with viral filtration and buffer exchange. As demonstrated herein, AetfeXia-hfio protein was purified to a purity bffo-fi8% as deternnoed by size exclusion chromatography and >£5% as determined by SDS FACIE. This level of purity was suffiefeht to achieve desirable effects on hone in mice and an acceptable safety profile In mice, rats and non-human primates.

In another embodiment, aihsion gene coding for a purification leader seeprenee, such as a poIy-(His)teater©k:inase cleavage site serptenee at. the M-terminus ofifoe desiredporrionof dm reeomhlnantAeiferia polypeptide, can sdlow purification of foe expressed fusion protein by affinity chromatography using a Np metal resin. The purification leader sequence can then be subsequently removed by treatment with enterokinasc to provide the purified ActRlla polypeptide (c.g., see Hochuii et aL, (198?) <1 C^{m}0ϋgnφh)f 4^1:177; ^NAS UBd 88:8972).

Techniques for making fusion genes are well known. Issentiaily^lbe joining of various BMA fragments coding for different polypeptide sequences is performed In accordance vrith conventional teclmlquesceniploying hlnni-ended or stagger-ended termini for ligation, msiriaidn enzyme digestion to provide for appropriate iermlnL as appropriate* a&amp;aline phosphatase treatment to awld uRdesirabifejoMmg, and..en^ms$© ligation. In another embodiment, the fusion gene can be syntoeslned DMA syomesi wan. Alternatively, FCR amplification of gene fragments can be carried out using anehor printers which give rise to compleoieotary overhangs between two conseentwe gene feagments which can subsequently be .'annealed, to generate a chimeric gene sequence (see. %y .example,. Current JPiwfamis to Mikcuiat Mmlag}^ eds. Ausuhei efaL Joint Wiley &amp; Sons: 1992). 4. Alternative Activin and AciRIIa Antagonists

The data presenfed herein demon Aratoa that aotogonlstopf aetlvIn^ActEIIa. signaling can he used to pfctPhte bone gtowth and bone mutemilgMinh, Aitobngh soluble ActRUa polypeptides* and particularly Aetrlia-Fo, are preierfed antagonists, and altbougb syeh ainagonists may afifeet hoite through a niachanpm otherdian aeiivin antagopistu (e.g.;3 activist inhibition may be an Indicator of the tbndeney of an ^iss;^.m<>leo0libsi members of the TGDheta superfamily, and such collective inhibition may lead to the desired ef&amp;ot on .bone), other are expected to be useful, Including and-aetivin (e.g.5 A, B, C or E) antibodies. anti-ActRIIa andhodies, antisense, RNAt or riborysne nucleic acids that inhibit the production of ActRUa and otoer htMbitorspf aetivin or AciRIfa, pardcaiarfy those that disrupt acts sdu-AetRIIa binding.

An antibody that M specificaily reaetwe With an AoSIIa polypeptide (e.g,s a soluble AcfEIia polypeptide and which eitoer Mads competitively to ligand with toe AotRIIa polypeptide tn otherwise inhibits AetRlltotoediatod signaling may be used as an antagonist of ActRIIa polypeptide activities- likewise, an antibody tost: is specifically reactive wlth an activist Apolypeptlde and which disrupts ActRUa binding: may boused as astiasdagonist

By using immunogens derived: front an ActRUa polypeptide or atractlvin polypeptide, antl-proteto/anti^peptide antisera or monoclonal antibodies can; be made by standard protocols (see, for example*: Antibodies: A Laboratory Manual ed. by

Harlow: and Lane {Cold Spring Harbor Press; 1988)}. A mammal, suck as a mouse, a hamster or rabbit can be immunised with m. imnmnogenie flam of tbs AetRIIa polypeptide, an antigenic fragment wliieh is capable of eliciting an antibody :response,:or a fission protein. Tebkfrques for conferring mmnmogcnleity on a protein or peptide include conjugation to carriers or other mchoiqnes well known in the art, An immunogenic portion of an AetRIIa or activio polypeptide can fee administered In the presence of adjuvant. Tbs progress of immunization can fed: moniioredhy detection of antibody titers in plasma or serum. Standard EI.ISA: or other iaunuooassay s can be used with the immunogen as antigen to assess the levels of antibodies.

Following luimnnlZatiou of an animal with an antigenic preparation ofan AetRIIa: polypeptidCj antisera can: be obtained und, if desired, polyclonal antibodies can he isolated fi'om: ihe scmin:. ϊο produce monoclonal; antibodies, antibody -producing cells (lymphocytes) can he harvested: from an hnmunfred animal and fused by: standard: somatic cell fission procedures with itnmertai iaing ceils such as myeloma: eellsto yield hyhridoma cells. Suck techniques are well known in dm art, and include, for example, the hyhridoma technique ^igmally developed fey Kohler andMlistei», (1975) Mature, 256: 495--497¾ the humaKB eeii hyfendomatselmique (Koxfear et ai, (1983) immunfrlegy Today, 4:72¾ and the EBMiyimidoma technique to produce fiumao monoclonal aodbodmsfCoie et aL, (19S5) Monoclonal Antibodies and-Cancer Therapy, Alan R. lissi IPo. pp, 71-96), Hybridoma cells can fee screened immunochemical^ tor pmduction rAantifiodies speoifically reactive with an AetRIIa polypeptide and monoclonal antibodies isolated from a onlture comprising such hybridoma cells.

The term “antibody” as used herein is intended to include fragments thereof which are also -specifically reactive with a subject polypeptide. Antibodies can be fragmented using conventional techniques and the fra®tmnts screened fer utility in the same manner as described above tor whole antibodies. For example, 1¾¾¾% fragments can fee generated by treating antibody with pepsin. The resulting Ffab)o fragment can he treated to reduce disulfide bridges to produce Fab fragments. The antibody of the present invention Is further Intended to Include bispeeifk·, single- chain,chimeric, inyimfked and idly human molecules having affinity lor m Acfiffia or aciivm polypeptide conferred antibody.

An antibody may firrther comprise a 1aM4®i^bidd:^«ir^'M€abfo: to he detected (e.|.,..-tbe label can bo a radioisotope, fiooreseent oorapomjd, enzymeor &amp;m$m&amp; co-factor).

In certain embodiments, the antibody is a reeorobirkni antibody, which term encompasses soy antibody generated In part by techniques of molecular biology, including GDffigtufted or chimeric antibodies* hbmas or other antibodies assembled fiom librnry'seiected antibody domains, single chain antibodies and single: domain antibddlesXe,g., human Vh proteins br camelid Vpi proteins), in certain embodiments, is-)* monoclonal antibody, and In certain embodiments, the invention mabes available'methods tbr generating novel antibodies. For example, a method for generatmg a monoclonal antibody that binds speolSealiy to an ActRIIa polypeptide or actlvln polypeptide :may comprise administering to a mouse an amount of an immunogenic composition comprising the: antigen polypeptide effective to stimukts a detectableimmune response, obtaining antibody-producing cells (e.g,, celts from the spleen) from the amuse and timing the antibody-producing ceils with myeloma cells to-obtain amihody-produciug hybridomas, and testing the adtibody-prodocing hybridomas id identify a hybridoma that produces a monocokmai antibody that binds specifically to the antigen. Once obtained, a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridom a-dcrived cell s produce &amp;e monoclonal antibody that binds specifically 10 the antigen. The monocional antibody may he purified from: the cell culture.

The adjective “specifically reactive with” as used in reference to an antibody is ibtenned to nmah, as is gencially understood in fi® art, that the antibody is sufficiently selective between the antigen of interest (mg., an ActRIfa pdlypcftide) and other antigens that are not of interest that the antibody is useful tor, at minimum, deteciffigthe presence of the antipm of hitemst in a particular type of biological sample. In certain methods employing the antibody, such as therapeutic applications, a higher degree of specificity in funding may be desirable. Monoclonal antibodies generally have a greater tendency (as compared to polyclonal antibodies) to disemBmate eHedmdy betwee» Jhe desired aniigsss^sd erossdmotmg polypeptides. One characteristic that influences the specificity of an andhodymntigen interaction is the affinity of the amlbody for the antigen. Although the /desired speeMdiy may be reached with a range of diBbreat: affinities, generally .pretoed antibodies will have m affinity' (a. dissociahmi constant) of about HP* 10'';, IG‘\ 10* or less, Given the eytraordinarlljrtight binding: between. aetMa and: ActRila, it is expected thiat a aeiitmliMng antAacrivla or anti-AetRIIaaBtihody word'd generally have a dissociation consianfof 10'5lj or less. la additlon/the teeimiques used to, semen antibodies k order to identic a desirable antibody ai;ay influence flm properties of the antibody obtained, F|r example, if an antibody is to be ased fbr bidding an antigen in. solution, it may be desirable to test solution binding. A ‘variety of different tohniqass areavailabie for testing interaction between antibodies and: antigens to identify particularly desirable antibodies, Snchfecbphpes molods EOSAs, surface plasmon msonanee binding assays fe.g,/the Biaeca^^ binding assay. Bisects AB, Uppsala, Sweden), sandwich assays (e.g,, ke paramagnetic bead system of ICMM Intematlonai, Inc,, Gai&amp;ersburg, Maryland), western blots? .hvmntnoprecipitatiort assays, and imrnunohistochemistry. vEtctop!e$^f^^g^e^\df:kici^acM compounds that are acbvm or A<^^C$#^o|ikk''kP|tke mBisenae nncieic acids, RNAi constructs and catalytic nucleic acid constructs, A rmclete acid compound may b© single or double stranded. A ddfhle stranded emnpound may also include regions of ovefhartg nr non-complementarity, where cam or the other of the strands is single straaded. A single stranded oompoaad may mo! ode regions of self-complmnentarity, meaning that the compound forms a so-called ffiaiiphT or ‘‘stem-loop” Aructure, with a region of double belies! structure. A nucleic acid compound may comprise a nucleotide sequence that is complementary to a region consisting of no more tat 1000, no more than 50¾ no more than 250. no more than 100 or no more than 50,35,30,25, 22,20 or l i nncleotides of the Mi-length AetBIia nucleic acid sequence or activin pA or actfvk pB nucleic add serpmncc, The region of complementarity will preferably be at least 8 nucleotides, and optionally at least 10 or at least 15 nucleotides, and ophonaiiy between 15 and 25 nucleotides. A region of ouMplemenlaniy may fall with® gmlfttro», a coding sequence otmmonboding' sequence of the target tmnscaipt, such as the ceiling sequence potfiorn 0e«arai!ys a sncleicdeid compound' Mil: have a length of about 8 to about S9S|hneleoideum base pains la length, and optionally the length will be about 14 bo abqidiCt tuibleotides, A nuclefe: aeidmay be a DMA (particularly Ibr use as anlaslisesse), RMA or fiMA:l3^Aiybi1d- Any ope snaud may iuclbde a mixture οί ΠΙ^Α ajtd RNA,as weliasimodiied forisathat cannot feadpy be classified as aiher DMA or ENA, Likewise, a: double strauded composed stay be DNAiDNA, ΙΟΝΑί&amp;ΗΑ. or KNAiKNA, and: any one strand may also molude a mixture of 0ΗΑ iUnddSHAi : as well aamodlfied tens that cannot readily be classified as eithet' OblAor 'MMA, A nuoidic acid compound may Include any of a variety of modifications, : inehsdirsg one or modifications to the backbone (tbe so gas-plmspbate portion in a satural nucleic acid, iadiudtttg intenmcleotide linkages) or dm: base pardon (¾ purine or pyrimidine portion of a natural: nucleic acid). An aitisense mAalalc aeidcompound will preferably have a length: of about 15 to about 30 nucleotides and will often contain oue :or mofe:modifieatious: to improve oharaeterisfes such :as: stability In the serum, in a cell or in a place where the compound Is likely to be deitv^Bd, such as die stomach In the case of orally deiivsred componnds and 8» 18¾ lor inhaled emspousds. In the ease of an fiMAl construct, the strand esiupiguteumry to the target transcript will generally iiereoft The other strmid may be R.NA, DNA or any other variation. The duplex portion of double stranded or single stranded ^hairpin” RNAi construct will preferably have a length of 18 to 40 nucleotides in length and optionally about 21 f.o 23 ducisoddes in length, so long as it serves as a Dicer substrate. Catalytic or enKymatie nucidlc acids may be ribozymes or DMA enzymes and may also contain modified ibr/ns. Nucleic acid compoun ds may inhi bit expression of the target by about 50%, 75%, 90% or store when OQ^cicd^^:C^k'ipid#::^yMQlcg)cal condidous arid at a concentmfios where a nonsense or sense control has Ififie or no effect, Pmferred epnoenftations for testing the «fleet of nucleic acid compounds are 1, 5 and 10 nuctornolav. Nucleic acid compounds .payier example, bone growth and mineral featinn. S, Screening Assays in certain assets, the pffesehiJwvefitlon'T^ktsS'to the use of ActRIIs polypeptides (e;g., soluble AetRRa polypeptides) and aetlvis polypeptides to identify compounds (agents) which are agonist m antagonists of the aetivin-ActRlIa slgnsling..pathway. Compounds identified through· this screening esn. be tested id assess their ability to. modulate bone growth, or .nnneraiizaiihn In vitro. Optionally,: these compounds can farther be tested in animal models to assess their ability to modulate issbegtowth ip vivo,

Thereare numerous approaches to soreenttig for thempeatic agents for. mo#ktiag#s$ue growth by targeting aciivm and AotRXla polypeptides. In eertal» embodlmeats, high -tferoughput screening of compounds can be carried out to idetrti% agenfe ibm pednrb activin or ActRIfenediated eileeis on hone. In certain emhodimerits^ the assay k carried out to screes and identify compounds that speoffically inbifeit or reduce binding of an ActRiia polypeptide to activin, AlWrnahveiy, the assay can he us0d'.to'::lte.tify'€tpt»p€te#:th3£ enhance binding of an ActRIIa polypepdde to actiyim In a further· embodiment, the compounds can he identified by their ability to Interact with an aetivln or AbtRlfa polypeptide. A variety of assay Ihpnats will sufice and, In lighipf the present disclosure, those not expressly described herein will nevertheless be comprehended by one of ordinary shill In tire art. Asdeseribed herein, the feat compounds (agents) of the invention may be. created;by any combinatorial ebemleal method. Afternativefy, the subject compounds may be satoraliy occurring biomolecules: synthesized It vfyo or in vitro. Compounds (agents) to beiested for their ability to act as moduMor&amp;of tissue growth caU: be produced, tor example, by bacteria, yeast, plank or other organisms (mg., natural produetsjyproduced chemically (e,gi4 small molecules,: including peptidomlmetics), or prodneed reoombmantiy<: Tbst compounds contemplated by the present invention Include non-peptidyi organic molecules, peptides, notypcptidesrpptdomimetlos, suprs, hormones, and nucleic acid molecules, In a specific embodiment, the test agent Is a small Organic moleeule having a molecular weight of less than about 2,000 daftons.

The test compounds of foe im*ntloo can be provided as single, discrete entities, or pmvided fodibraries of greater complexity, such as made Iw comhinatodal chemistry, These iihrariescan comprise, for example, alcohols, attcyl halides, amines, ^ji^^fe^-aldehyde^^tiiss^and other classes of organic compounds. Presentation ofte^^mpoundsio^he'te^'^^^a^^i bein either an isolated form or as mixtures of compounds, especially in initial screening steps, C)|)dnnai]ys foe compounds may be optionally detfoatized wifo other compounds and ham iteriyatizing groups that facilitate isolation of foe compoiisds, Nondimiting examples of derlvathdug groups include biotin, Snoreseeln. digoxygsnm, green fluorescent protehL isotopes, polyhistkilne, niagneiie heads, glutathione S transferase |GST), phoix^activatMe crosslinkers or any combinations thereof

In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are perfected in celMree systems, such as may he derived with purified or setmk purified proteins, are often preferred as feimary” semens to feat they-can he generated to permit rapid development and relatively easy detection of an aheratlon in a molecular target which Is mediated by a test compound, iforeovep the effects of cellular toxicity or bloavai lability: of foe test compound can be generally ignored In the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as: may be manifest in an alteration: of binding affinity between an AciRIia polypeptide: and activln.

Merely to illustrate, in an exemplary screening assay of the present invention, foe compound of interest is confected, with: an isolated and purified AciRIia polypeptide which Is: ordinarily; capable of binding to activla, To the mixture-of the compound and ActMa polypeptide is then added a composition containing an AcfRXXa ligand, Detection and quantification of AfeXOXafeetivin complexes provides a means for determining the compound’s efficacy at inhihiting for potentiating) complex formation between the ActRIIa polypeptide and act! vim The efficacy of foe compound can :be assessed by generating dose response curves from data obtained using various concentrations of foe test compound. Moreover, a control assay can also Ife performed to provide a baseline for comparison. For example, in a control assays aetivmls&amp;dded: to a composite containing the::AetM^ M&amp; the formation of AeiRlIa/acilvm complex is quantitated in the: absence offoetesi coispi?a»d. It will be understood that, m general, the order in "Which: tbs reactants may fee admixed cap toe varied, and can he: admixed sinmlim:eou$ly, Moreover, la place:of puriifed protemss cellidar extracts and lysates may : be used to reader a suitable cell-tree assay system.

Complex formation be^ift-'Sje-.AdtRIIa jKjiy|!!epde:.mid.'activin may be detected fey a variety of tecim%fe#> FofmSt&amp;uee, modittaiiod: of the formation of complexes can fee quMt|^tsa|:'«sih^,'for exSgppie, detediably fafeeied protems snefe as : mdiolatoeled i4C or 41), ilimrescentfy labeled (e.g,, FiTC), or enzymatically labeled AetRHa polypeptide or acivlfe, by Immunoassay, or by eh rornatogmpMo detection. la certain enfoodimenfcs, ibe present .mvention edaesmpigtes fee use of tlnorescence polarization assays arid fluorescence resonance energy transfer (FRET)1 assays in measming: either directly or mdlmcdyS: tie degree of interaction between an. AelRTIa.polypeptide and Its binding protein, Further, other modes of detection, stteh ic^iwaveguides (F0F Fublieation W© 96/26432 andlhS,

Fat. Npv 5,677,196), surface piasmon resonance (SPE), surface eharp sensors, and surface force sensors, are compatible with many embodiments of the in vention.

Moreover, the present invention eoRtempktes the use of an Interaction trap assay; also known as the *Hwo hybrid assay,- for ident%in|; agents that disrupt or pofoudam interaction between m AetRIIa polypeptide and its binding protein. See for example, IkS, Fat. No, 5,283,3.17; Zervos et al. (1993) Ceil 72:223-232; Madura et at, (1993) 1 Biol Cfeem 258:1204642054; Bartel el ah (1993) Biotechniques 14:920-924; and Iwabuchs et ah (1993) Oncogene 8:16934696), In a speelBe embodiment the present invention contemplates the use Of reverse two hybrid systems to identity compounds (teg., small molecules or peptides) &amp;at dissociate interactions between an ActRIla polypeptide and Its binding:protein. See for example, Vidal and Ingrain, (1999) NiiSleic Acids Res :27:9l.949;Vidai and Legraln,: (1999) Trends Bioteehnol 17:374-81: andvtkSfPai.Moe, 5,525,490; .5,955,280;: and 5,965,3 68. ίο certain enrbddhnents, the subject cosapouMs are icietsiiled bv their ability to interact with an AetRife or antivia polypeptidb-of ^..tnyenfi^ The Interaction between tire compound assd Ate AetFIia or activin polypeptide may be covalent or : «onwovaient. For example^ such Ihtsfcaction <^>:fre. ipmteia.level using. I» vitrp bioelheip led methods, including phofercmsslmkiog, radiolabeled ligand binding, and affinity dUOTaiogmphy (Jakoby WB etal,5 1074, Methods fe Bpzymoingy 46: 1). In certain eases.the compounds may be screened to a mechanism based assay, such as no assay to detect compounds which hindto: an activin or ActlJla polypeptide^ This may ioelude a spIM ph^e or fldid phase binding «vent: Alternatively, the gens encoding im activia or AotRJto polypeptide can be transfected with a reporter system {e<gi, p^lastosidase, ludieiase,. or green fluorescent protein) library preferably by a high throughput screening or with huhyidani members of the library. Other mechanism feia^dinhdiug -assaysmay be used* h»· example, binding assays which; defect changes In free energy; Binding assap can. be performed with the target fixed to a well bead or chip or captured by anitnmobiifeed antibody or resolved by capillary eiectrophoresis. The bound compounds may be detected usually usmg colorimetric or fluorescence or surfece ptasmon resonance.

In certain aspects, fee present Invention provides mefeods and agents fer roodnlatlhg (stimulating or inhibirijrg) bone fermation and increasing bone mass, thetefee*. any compound· identified :Oaa-%:fesfed in whole cells or tissues. In vitro or in vivo, to confirm· their ability to; modulate bone growth or mineralization. Various methods known in the art can be utilized for this purpose.

For example, the effect of the: Acts.IIU: or aedvih polypeptides or test eotnpou.u.ds: on bone or caTtiiage growth can be detennihfed by measuring iifeoetioh ofMsx2: or differentiation of osteoprpgenitor cells into osteoblasts in cell based1 assays (see. e,g>,: Dalniski et ai, Hat Cfenet. 2001,27(I):84«8;: HIno et al;s Front B foset 2004,0; 1520*9), Another example of ccil-based; assays includes analyzing the Qsfeogbnic activity of the subject ActRIIa or aodvihpdiype|tides and test: compounds: in mesenchymal progenitor and osteoblastic ceils, To: illustrate,. . r^mbhiaht .adebdyiresgis: «scpressiitg an activin or AetRIia polypeptide can be constructed to inieet pjuripotent mesenchymal progenitor C3HI0X1/2 ceils. preosteoblastic C2C12 c@H% and osteoblastic TB-SS oeiIs,: Osteogenic activity is then deteropnedi: by measuring the/induetion of alkaline phosphatase, osteocalcin, and matrix mreeralfeatiQn (see* e,g„ Chengei al>, J boas Joint Snrg Am. 2003, M~ A(S): 1544*52).

The present mventlon also contemplates la vivo assays to measure bone or caMlagp growth,: For example. Namkrmg-Matthai et ah, Bone, 28:80-84 (2001) discloses a rat osteoporotic mode! io which bone repair during the early period after Irecture is studied, Molecular Biology, 68; .197- 2t)2 (1999) also discloses a rat osteoporotic model in which bone repair during the late period after fracture is studied. Andersson et at, J, foidocrlneS, 170:539^537 describe a mouse osteoporosis model in which mice are OMrieefoptieed., which causes the mice to lose substantial bone mineral content and'bone mineral density,: with the tjsbeeulhr hone losing roughly 50% of bone mineral density. Bone density· could he jpcraased ia the owkcfonifeed. mice by administration of factors such as pafaftuftbid hPruionei In certain aspects, the present invention .snakes: use: of frUCfMre healing: assays font are kaown in the art. These assays Include fraemre msalysis, and biomechameal analysis, which are described in, for example, tIS. Bat. bto. 6,521,750, which la Incorporated by re In its entirety for its disclosure of experimeutal protocols for causing as well as measuring the extent, of fractures, andthe repair process.

In certain embodiments, activio-ActRiia antagonists (e.g,, AcrRIIa polypeptides) of the present invention can Be used for treating or preventing a disease or cotfoitkm that is associated withbone damage, whether. e.g., through breakage, loss or denfruemfeiion.. In certain provides methods of Ireatmg nr preventing bone damage In an indl yidual in need foereofthrough administering to the individual a therapeutically effective amount of an aetlvin^tetRIla antapuisf, p&amp;rdeni&amp;rly an ActRIla polypeptide, in certain embodlnmnts, the presere invmUi on provides methods of promoting bone growth or mlnerahzatlou iu an individual In need thereof through mfministering to the individual a fterapeuris&amp;Ily effective amount of an aciivinwteiEIIa antagonist, particularly an ActRHa preferably aimed at tfer&amp;peutfc and 'prophylactic treatments of animals, and mors preferably,; hnaaaus, fa eejtaliremhodiiaestSj. fee disclosure provides for the use of aotifenwfetlOIa antagonists Cpsrtiealarly soluble AetRIla polypeptides and nenirfeking anrihocfe targeted to adftvi)SW-:lfcC^na);:&amp;r<he:-fep^amt of disorders associated wub low bone density or decreased bone strength.

As used herein, a therapeutic that ;ipreveri|s5i a disorder or dobdltioh yefes fo a compound that, in. a statistical sample, reduces the occurrence of the disOider or condition fa the treated sample relative to an untreated control sample, or delays the onset or reduces the severi ty of one or more symptoms of the disorder or condition relative to the ontfesfed control sample. The term fereatmg” as nded hefein Includes prophylaxis ofthe named condition or amelioration or eitmiraiiion of fee condMoa once It has been established. In either case, prevention or tmatmehtmaylm discerned in the diagnosis provided by a physician and the intended msnlt of administration of the therapedtic agent,

The disclosure provides:: methods of inducing hone and/or cartilage formation, preventing bone loss, increasing hone mhieralfeatlon or preventing the demineraikation of hone,; For example, the subject aeftyit^AetEIIaantagohlsishave application hi treating osteoporosis and the healing of bone fractures and cartilage defects in humans and other animals, AetRIfa or scdvlo polypeptides: may heuseftd la patients: that are diagnosed, with subclutfeal low bode density, as a: protective measure against the development of osteoporosis.

In one specific embodiment, methods and compositions of the present invention may find medical utility In the healing of bone Iraetdres andcartilage defects m humans and other animals, The.subject methods and compositions may also have; prophylactic use In closed as well as open fracture: reduction mid also. In the improved fixation of artificial joists, De novo bone formation Induced by aft osteogenic agent contributes to the repair of congenital, iramsarinduced, or onooiogie resection ' induced, craniofecla! defects, and also is useful in cosmetic plastic surgery. in certain eases, the subject actiyln^ctRIIa amagonlsts may provide an environment to attract bone-toonlng: cells, stimulate: .growth #boae~ fenning colls or Induce diifemntiatian oi propostors of booe-formiog ceils. Activin-Aeifeila antagonists ofthe invention may also be useful in the .treshaeat of osteoporosis.

Methods and compositions of tire Invention can be applied to conditions chatacterked by or causing bone loss, such' as osteoporosis (inchidhig seomidary osteoporosis), hyperparathyroidisms Cushing’s disease., Paget’s disease, ttorrotoxieosis, chronic diarrheal state or nmiabsorplion, renai tubular acidosis. Or anorexia nervosa.

Osteoporosis may be caused by, or associated with, various Isctors, Being female, pariionlariy a post-menopausal female, having a low body weight, and leading a sedentary lifestyle are ail risk feciors for osteoporosis (loss of hone mineral density* leading to fracture risk). Persons having any of the tallowing profiles may be candidates for Ematment wife an AmRIIa antagonist: a post-menopausal woman and not taking estrogen or other hormone replacement therapy; a person with a persoml or maternal history of bip feaciare or smoking; a postmenopausal woman who Is tall (oyer 5 feel 7 inches) or thin (less than 125 pounds); a man with clinical conditions associated with bone lass; a person using medications that are known to cause bone toss, toelodmg corticosteroidscsuch as Prednisone™, various ami-seiaure; medications such as Dllantm™ and certain: barbiturates, or high-dose thyroid replacement drugs; a person having type I diabetes, li ver disease, kidney disease or a family history of osteoporosis; a person bavmg high bone turnover (e,g:,, excessi ve collagen ih urine samples);: a person, with a thyroid condition, such as hyperthyroidism ; a person who has experienced a fecture after only mild trauma; a person who has had xway evidence .of vertebral imetore or ether signs of osteoporosis.

As noted above* osteoporosis can also result as acondition associated with another disorder or from the use of certain medications. Osteoporosis resulting from drugs or another medical condition is known as secondary Cfsteopdrosis. In a condition known as Cushing’s disease, the excess amount of cortisol produced by the body results-1«. osteoporosis and i acteres. The most common medications associated with secondary osteoporosis: are the corticosteroids, a class df dregs, that '«cfclike. ©cM’tliSols.-a.p*<^iiped:.i#S3^i'|Sy· toy· fee adrenal glands. Although, adequate levels of thyroid hormones; (which ate produced by the thyroid gland) he^ed'-for sfeleton, excess thyroid hormone cat) decrease bone mass over lime. Antacids that eordaln alna&amp; um can load to hone: loss when taken in high doses fey peopl^lii^j^^ydhiOTS, particularly those undergoing dialysis, Other medications that can cause secondary osteoporosis incinde f^eoytoln (Dliadtlh}: and: barbiturates that are: used to prevent seizures; methotrexate (Rheumatrex; Itnmuhex, Folejt-S»ir- some forms of arthritis, cancer, and immune disorders:; cyclosporine: (Sandhnnntne, Meoral), a drug used to treat some autoimmune diseases and to suppress the immune system in organ transplant patients; luteinizing hormosfo^ agonists (Lupron, Zoladex), used to treat prostate cancer and endmnetrlosis; heparin :{Caicipariae9 Liquaemin);, an aoilclotthig medication; and cimies^inmme ifQiteairan} and colestipol {Colestid}, used to treat high cholesterol. Bdhe loss: respiting from cancer therapy is widely recognized and termed cancer therapy induced: hone:loss (CTIBL). Bone metasiases cm create cavities indie bone that may be eerremed fey treatment with activin-AntRXia antagonists, in a preferred embodiment activm-AetRHa ^tagonis^i^r&amp;ately'.a soluble AetRIIa, disclosed Serein may fee used In cancer patients, Batlents having certain tumors (e.g. prostate, breast, multiple myeloma or any tumor eansing hyperpamdrymluism) are at high risk for hone loss duo to tumofdndueed bene loss as well as hone metasiases and therapeutic agents. Such patients may fee treated with activin-AeiRlIa ahiagonisis even in the absence of evidence of hooe loss or bone meiastases. Patients may also he monitored for evidence of bone loss or ixme metaspsesj and may belre&amp;md wkh activin-ActBIIa antagonists in the eyeni that indicators suggest: an; ifeercased risk. ffenerallyj DEXA. scans arc employed to assess changes In bone density, while indicators of hone remodeling may be used to assess the likelihood, of hone metastascs, Serum markets may he memrored. Bone specific·: alkaline phosphatase (S SAP) Is an enzyme that is present In osteoblasts. Blood levels of BSAR are::increased in patients with bone metastasis and either conditions that resuiiin increased bone remodeling. Osteocalcin and prooollagen peptides are also associated with bone:formtek>n add bone thfeastases. foemases I» B SAP ha# been detected ΐϊί:0^^·ψ^ι!1ι^·^^φφίβ caused by prostate cauoer, and to a lesser degree, lQ.hcme:t8^$*^';|^.,i(r<5isi· cancer, Bone hforphogeneiio Protein-7 {BMP-7}· levels are high. in: prostate oaneer feat has metastasized to bone, bat oot la hose aietastaseS: dqs to bladder, skin, liver, or long cancer. Type Ϊ Carbo^-termmai teiopeptide (ICTP) Is a crosslink: found :m collagen: feat is formed during to the resorption of bone, Hides bone is constantly being broken down and reformed, 1STP will be found throughout the body. However, at fee site of bone metastasis, the level will be significantly higher than in an area of nonMi bone. ICTP lias been found in high levels in bone metastasis due to'prostate, lung, and breast cancer. Ahofeer collagen crosslink, Type IN-tenninat teiopeptide (NTx), is predueed along with ICTP dtsrmg hone turnover. The amount of NTx Is Increased in bone metestasls caused by many: different types of cancer iaolndlug lung, prostate* and break dancer. Also, the levels of Mite Increase with the progression of the bone metastasis. Thmfo^;..^Iihail^Ci&amp;#-be ttsed to both; detect metastasis as well as measure the extent of feeVdisease, Other markers of resorption Inc lude: pyridinolme . and deoxypyndmoline. Any iodfease In resorption markers1 :m markets of bone metastases indicate foe need for acii vin-AciRIia; antagonist therapy in a patfest,

Amvin-Aetllla antagonists may be conjohitiy administered with other pharmaceutical agents. Conjoint admlnitersiion ntay be uecofeplishod % admlnistrat ion of a .single. oo»formnlations by simultaneous administration or % adnhnistration at separate times. Actlvm-ActMla amagonisismay be parlieuMly advantageous if afenlnjstered with other bone-active agents. A patient may bens® from conjointly: receiving aeiviir^dRila antagonist and teking calcium snpplemeptst vitaraib D, appropriate exercise and/or, m some cases, other medication, Examples of other: medications Incude, feisphosphomtes (aiendronate, Ihandmhafe hnd risedroaate}, oatelfanlp,, estrogens, parathyroid hormone and raloxifene, ibandronate and risedronate), eaickohip,:estro:gen:S:;asfe ralokifone;affect the bone remodeling cycle and are okssifted as unti-resorptive medications:. Bone remodeling consists of two distfeet stages; bemsirosorption and bone formation, Anthresorptlve med-ktelons slow or stop fee boue-resorblng pardon of fee bone-remodeling cycle bid do not plow fee bmk#>Hning portion of the: cycle. As a result,, newformation continues atagreater rate than. bene resorption, and, bone density m»y increase over time. Tmparatide, a lor*» of parathyroid; hormone, increases ihemteof bone: formation Is the boae remodeling cycle, Alendronate Is. approved for both, the prevention (S mg per day or 35 rag. am® a weel;} end treatment pb mg: per day or 7CI: mg oneea week'> of postntenopausal osteoporosis; Akodronate reduces bone loss, increases bone density and reduces the risk of spine,. wrist and; Mp fractures. Alendronate also is approved far treatment of gkteaporiicoiddadnesd osteoporosis in men and women as a result of long-term use of these medications (he., prednisone and cortisone) and for the treatment of osteoporosis in men. Alendronate plus vitamin D Is approved tor the treatment of osteoporosis in posimcaopansaltwomen (7d mg once a week plus vitamin D), and tor treatment to improm hone mass in men with osteoporosis. Ihandronate Is approved for the prevention and imaiment of postmenopausal osteoporosis. Taken as acmee-a-month pill (150 rngk lbandronate should be taken oa the same day each month. Ibandronate reduces hone tcgsptncreascs bone density and reduces the risk of spine fractures. Kisedronate is approved for the prevention and treatment of postmenopausal osteoporosis. Taken daily (5 mg dose) or weekly (35 mg dose or 35 mg dose with calcium). risedrooate slows hone ldS% increases bone density and reduces the riskof spine and non-spine fractures.. iisedronate also is approved for use by men and women to prevent and/ortreat glucocortieoid-indueed osteoporosis that results from long-term, use of these medications (ite>, prednisone or cortisone). Calcitonin k a naturally occurring hormone involved in calcium regulation and bone metabolism, in women who are more than S years beyond menopause, calcitonin slows bone loss,: inemases spinal bone density, and may relieve the pain associated wild hone fractures, Calcitonin reduces the risk of spinal fractures. Calcitonin is available as an infection (50-100'EJ daily) or nasal: Spray 000 IF daily). Estrogen therapy (EthilJormone therapy (HT) Is approved for the prevention of osteoporosis. ET has been shown to .reduce boneless* Increase bone density; in both the spine and kipyand reduce the risk of hip and: spfnaHractures in postmenopausal women, ET &amp; administered ntosi; commonly in the form of a pill or skin patch that delivers alow doseof approhlmately 0,3; ;rag: dally or astandard dose, of appfokimately· 0,525 mg daily and is effective ©yen when started alter age '70. When estrogen is taken alone, it oan increase a woman's risk of developing cancer of Ίο efcinatelliis risk, healthcare piovyerS''P^^@'^'^rmone'pOj^^ in replacement-therapy or HT) for those women who have an. Imaet uterus. ΕΊ7ΕΤ relieves menopause symptoms and has Been shown to have a beneficial effect on hone health. Side effects may mchnie vaghnti hfeeding^ breast fondeimess, mood disturbances and gallbladderdisease., Raloxifene, hO mg &amp; day* is approved for the prevention and tmahnent of postmenopausal osteoporosis. It is from aelass of drugs odhfo Sekmfive Estrogen Receptor ModuiatorspMfMs) that have been developed to provide the beneficial effects of estrogens without their potential disadvantages, Ifoloxifone increases bone mass and reduces the risk of spine fiaefomsrldata am not yet available to demonstrate that raloxifene can tedtice the risk of hip and other non» spine iraetutas, Teriparatide, a form of pamtliyroid hormoue, is approved for the treatment of dsfoeporosls in postmenopausal women and men who are at high risk for a fracture. This medication sthnulates new bone formation and significantly Increases bone minera! density. In postmenopausal women, fracture redaction was noted in the spinej: Mp, foot, ribs anil wrist, &amp; men, foaotureawduotion w noted In the spine, hut them were iasutfioient daia to evaluate iMcture'redueiion at other sites. Tenparafide is selfradministered as a dally injection for up to 24 months.

Is.certain embodiments,: activimAefMIa ahtagonlsis (e.g., AelRIla polypeptides} of the present invention are formulated: with a pharmaceutically acceptable earner. For example, an AetRIia polypeptide can. be administered alone· or as s:component of a pharmaceutical formulation therapeutic composl don), The subipet compounds may he formulated for administration in any convenient way for : use In human or vefrainary medicine*

In certain embodiments, the therapeutic method of foe invention includes administering foe ermiposfoao systemiealfA or iocaiiy as an implant or device. When administered, foe foetapeufie cOmpositimi for use irt this invention is, of course, in a pyrogen-free, physiologically foeeptafrfefount. ''Ofompentlesliy useful agents other than the: AetRlla antagonists which may also optionally be included'in the composition as described above, may be adniihi^it4-S3i»uiis»eo»Siy' or sequentially' with the subject· compounds (e.g,* AetRIla polypeptides); in the methods of the invention.

Typicalty* AofRlIa antagonists will be admimsierhd parentslly, Fhatmacebbeal compositions suitable for parenteral administration may comprise : one or more ActtMa polypeptides: ip comhinailon with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaquebus solutions, dispersions, suspensions or emulsions, or sterile powders which may be ·&amp;3ίρ:.$ΙΝ^ί.<? iry cetabte solutions or dispersions just prior to use, which haeteriosiats, solutes which render the the blood of the intended recipient or suspending or drfekeaing agents, Examples of suitable aqueons and nonaqueous carriers which may be entplpyed in tlm pharmaceutical compositions of the invenaon include water, etbahol, polyols (such as glycerol, propylene glyoo!, polyeifeylene glycol, and the Ike), and suitable mixtures thereof, vegetable Oils, such as olive oil, and isrjeetable organic esters, snob as ethyl oleats, Proper fluidity 15¾¾¼ m#ia^meil,,fbr example, by the use of coating materials, such as lecithin, by the: mmotenanae of the required particle size in the ease of dispersions, aim by the use of surfactants, :Fur^erf|he.qo^pqsf^#-'tw-.|j!e encapsulated or injected in siform for delivery to a target tissue pile (e,g.:!:bone). in, certain embodiments, compositions of the present invention: may include a matrix capable of delivering: one or more iherapeatic compounds (e.g., ActRSa polypeptides) to a target tissue siie(e,g,, bone), providing^ sWimture lor the developing tissue and: optimally capable of being resorbed into the: body:. For example, the matrix may provide slow release: of the ActRlla polypeptides. Such mdricesmray be formed of materials preseutly In use: ;for other Implanted medical applications.

The choice of matrix material is based op hfeompatibiSty, biodegradability, mechanical properties, cosmetic^appearance and jnter&amp;ee properties, lire particular application of the subject compositions wiii define the appropriate fbnnukdlon. Potential matrices for the compositions may be biodegradable and chemically defined ealo&amp;m sulfate, ycafeiymphospEaie, hydlteyhpatte poMacifo acid: and polyanhydridos, Other potential materials are biodegradable and biqfogiealiy well defined, snob as bone or dermal collagen, Further; matrices are comprised: of pare proteins pr extracellular matrix components, Other potential: matrices are:«pn-biodegradadieiasd chemically defined, such as sintered bydiOxyapati%: hioglass, alumiiiateSj or other ceramics, Matdoss may be comprised of eombloafionS: of any of fe above mentioned types of material, socb as polyteotie aeid and hydroxyapatite or eoIiagsn: and taealclompbosphate, The biocsramics may be altered in : composition, ;sde$t ** $» and processing to ate pore sme, partlcicaizej particle: shape, and biodegradablhty.

In certain etrihpdlmeohg methods of the Inyention can be administered; for orally, e,g., in the form of capteess cachets, pills,. tablets, lozenges (using a iliavored basis, usually sucrose and: acacia or tragaeamh), powders, granules, or as a: solution or a suspension in an aqueous or nanmquequs liquid, or as a», oilte-water or water·* Intel liquid emulsion, or as an elixir or syrup, or as pastilles (using an intehase, such as gelatin and glycerin, or sucrose and acacia) like, each eontainlng a predetermined amount of an agent as an active ingredient. An agent may also be administered as a bolus, electuary or paste.

In solid dosage forms for (capsules, tablets, pills, dragees, powders, grannies, and its like), one ormom therapeutic compounds of the present invention may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dfoalemm phosphate, and/or any of the following: (1) fillers or extenders,. glucose, rnannrtol, and/or silicic acid; ¢2) carboxymethyleelfolose, alginates, gelatin, poly vinyl pyrrolldone, sucrose, andfor acacia; (3) humecianis, such as glycerol; (4) disintegrate a^ote, such as agar-agar, calcium carbonate, potato or tapioca starch, aiginie ;api4..mfe^n:^lkates, and sodium carbonate; (5) solution retarding agents, such as pamffin; (fo absorption accelerators, such as rpstemary ammoninm compounds; (7) welting agents, such ns, for exampfe, eelyl nlcohol and glycerol monostearate; (8} absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lanryi sulfate^ and mixtures thereof; and ¢10) coloring agents. In the case of capsules, tablets: a&amp;t! pills, the pharmacytipal composlifoRS PTay also: comprise buffering; agents, Solid compositions of a similar type nmy also be employed as lactose or milk sugars,. as well as high molecular weight polyethylene glycols and the like.

Liquid db$^ feme lor oral adrmnistrafioh include pharmaceutically acceptable emidsimrS, ndctoemnlsions, solutions, suspensions., syrups, and elixirs.

In addition to the asdve:ingredieb|: the liquid dosage forms may contain...mart diluents eomasonly used:in the art, such as water or other solvents», solubilising agents and: emrdslflers,: such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, l,3~feuiyle«e glycol, oils (¾ particular,: cottonseed, grounds 0¾. coop gerat, olive, castor, and sesame oils),: glyceroh fertahydrt?furyl alcohol, polyethylene glycols sod fatty acid esters Besides: inert diluents, the oral compositions can also include attfuyauts such as wetting agents, emulsifying and suspending agenkr sweetening, Savoring, coloring, pmiuniing, and preservative agents..

Suspensions, m addition to the active compounds, may contain suspendiog agents such as ethoxyiated Isosfearyt alcohols, polyoxyethylene sorbitet, and sorbitan esters, mieroerystallhte cellulose, aluminum metahydroxide, bentonite,: agaroigar and tragacantii, and mixtures thereof,.

The compositions of the Invention may also contain adjuvants, such as preservatives, wetting agents, emulsify mg agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the· inclusion of various antlb&amp;O'teriai and antifungal agents, tor example, paraben, ehlorobutanoh phenol, sorbic acid, and the like. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the inje£tdbie'''pf^ assy be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

Tb ls understood that the dosage: regimen will be deteratmed by the attending physician considering various feetors which modify the action of the subject compounds of the Invention |s.g<, polypeptides). The %riou$ factors include, feet are not limited to, amotmi of bone weight desired to fee formed, the : degree of done density loss, the slfe of bone dai»ages tfee conditio» of the damaged bone, the patient's age, sex, anddiet, tte severity of any disease that may he contributing·to'hone less, time of adinmisttaiion, and other clinical: factors, dptionaiiyi the dosage may vary with; the; type of matrix used In the meonstftntlon and the types of compounds in die composition, The addition of other known growth factors to itasimai composition, may also affect the dosage. Frogressean be monitored hy periodic assessment of hone growth and/or repair, for example, Xmays (including BEXA), and tetracycline labeling.

Experiment wti^^ denmnstrated that effects offtotMIfc-Fo on hone are deteotafele dhen the compohnd is dosed at intervals and amounts saffioient to achievesemmeono^^ or greater, and serum levels of i pg/kg of 2 ggkg or igeafer are desfeahle for aofeieviag siguifioant effects on bone density and strength. Although there is no indication that higher doses of AetMfa-Bd are tmdesirabla due to side effects, dosing regimens may be designed to reach serum concentrations of between 0.2 and 15 p^lcg, and optionally between i and 5 pg/kg. In humans, sertnn levels of ffi,2 ggdtg may he achieved with a single dose of 0,1 rng/fcg or greater and serumieveis of 1 gg/fcgmay fee achieved with a single dose of D J tng/kgor greater. The observed serum half-life of the molecule is between about 2¾ and 3d days, substantially longer than most Fc fusion proteins, and thus a sustained effective serum level may be achieved, fe eaampfo, by dosing with (1,2-0;4 m|gkg on a weekly or biweekly basis, or higher doses may be used with, longer intervals between dosings. For example, doses of 1-3 mg/kg might be u§ed on a monthly or bimonthly basis, and the effect on bone may he sufficiently durable that dosing is necessary only once every 3,4,5, fe, 9,12 or mom months.

In certain embodiments, dm present invention also provides gene therapy for the in vivo production of AetRffa polypeptides, Such therapy would achieve its therapeutic effect fey introduction pi the AciRfla polynueleofde sequences into cells or tissues having; the disorders asliafed above. Delivery of AetRIIa polynucleotide sequences can be achieved using a recombinant expression vector stseh a$ a chimeric virus or a colloidal dispersion spt&amp;ttu Preferred for therapeutic delivery ofActRBa polynucleotide sequences i$ fee use of tainted lip&amp;$oxtm.

Various viral vectors which can he affixed for gene therapy astaughtiicreln Include adenovirus, herpes virus, vaecihia, or, preferably, an RHA vims such as a re&amp;ovirus. Preferably, the retrovhaPvecto? is a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene east be mserfod include, hut are not limited fo; Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (BaMuSV), murine mammary tumor viras and Rons &amp;rcoma Virus (RSV), A n.urnber of addition8i :mt5X>viral veetm^ can iheorporate multiple genes. All of these vectors can tramfer or meorporate a gene for a selectable marker so that, transduced cells can he identified and generated.

Retro viral vectors can be made target-specific by attaching, tor example, a sugar, a glycollpid, or a protein. Preferred targeting is

Those of skill in the art will recognfee feat specific polynucleotide sequences can be inserted info the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector containing foe AetRIla polynueieotfoe. la a preferred embodlmehi, the vector is targeted to bone or cartilage.

Alternatively, tissue culture ceils can be; directly nansleeied: with plasmids encoding the retroviral structural gP^s gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid, containing the genes of interests. The: resulting cells release /the: retroviral vector into the culture medium.

Another targeted delivery system· for ActRIla polynucleotides is a colfoidal dispersion system, GoIMdal dispersion systems hrclude mncromoleeuio completes, nanocapspies, microspheres, beads, and lipid-based systems meiiullhg oii-m-wafer emulsions, micelles, mixed micelles, and liposomes, The preferred colloidal; system of this invention iaaliposofee, Liposomes are artificial membrane vesicleswhich am useful as delivery Vehicles in vitro and in vivo. ENA, DMA and intact virions can be encapsulated wihin fee aqueous Interior and be delivered to cells in a biologically active form (see e,g.. Raley, et al.5 Trends Bfochem. Scl, 6:77, 1981). Methods for efficient gene ifonsfer using aJiposmne veidcle, are known in the art, see e.g<5 Mamrmos et ah* Bioicehniques,6:d82,19M, The composition of the liposome Is usually a combination of phosphollpMs^ usually in ccanbinahon wil steroids, especially ctelesteraL Other phospholipids or other lipids may also he used- The physioaftdiamelsdsdes of liposomes depend op,pH, ionic strength, and the presence of divalent cations,

Examples of lipids useMln liposome predpedou include phosphatidyl eohipounds, such as phospiatldylglyoerois, phosphatiilylclK>liE&amp;, phosphatidylseme, phosphmidylethanolamine, spfnngolipids, oerebrosldeSi and gangilosides.

Illustrative phospholipids include egg phosphatidylcholine, dipaimitoylphosphaiidylcholioe, and disieatpyl|dmsphatidy!ehoiiBe, The targeting of liposomes is also possible based on, for·example, organyspeeijMfy, cell-specificity* and mganejie-speoiriehy and is teown in the art

EXBMHdFlC ATION fFhe invention now being generally described, it will fee more readily: understood by reference to the Allowing eMmples, which are included merely for purposes of lilustraifori of ceitain embodiments and embodiments of the present Inven&amp;m, and are pot intended to limit the invention.

Example 1: AotMia-Fc Fusion Proteins

Appiteahts constructed a soluble ActRIfa fusion protein feat has the exhacdlhiihr domain of human AetEMai&amp;^ihsvahimran'^ir:mdu$e Pc domain with a minimal linker in between, 1¼ constructs are referred to as ActRIIa-hPc and AetBIIa^mPc, respectively,

ActRIla-hPe is Shown below as purified fk>m CfiCi cell lines (S1Q ID NO: /}: iL6KSlTIX3BCMNA^^EiaElNQTGVEPCYO0KDKRKHOPA:TWiSNiSGSi

IlVR^GC^EDPmCYDRTDGTpKmSPEVYEeeilt^MGNiKFSYTPEMEV

Tt)FrSNPYTEKPFiYlGGTflTGEPCPAEHEEGGPSYFEFPPKPKDTEMlSR:IPEY

TCVYYDYglMEEVlfFNWY¥DGVEYIMAKlEmEOYMSTYEYVSYi;YYL aOPWGNGE^YRGK¥SNKAlPYPffiKTISKAKD0PlEPO¥¥TI,PFSR]isMTI·

N(')VSl i'('llVv?K(.TFY?$DIAVBWESNGOFENhrYIONlIyPV^I.X>SI>GSFFLYSKLrV DKSRWOOGNYFSC;SYMIffiALHNM7X>KSLSLSFGK

The AetRIId^hFe- and Acdtife-mFc proteins were ©iprsssed in GHO cell ikies. Hires different leader sequences were considered: (!) Honey bee meflitm (BBMQ; MEM^HMOTMVVYISYIYA (SEQ ID NO: 8) (p) Tlssde PSasmhQgpt Activator (TPA|· M0AMIOi£iLGeYTydCGA VPYSB (SEQ ID NO; 9} (Hi) Hbrive:; MGAΑΑΏ^AFAYFLISG8SGA (SEQ ID NO: 10).-

The selected fbtm employs the TPA leader and has the following improeessed amhtff aGd sequence:

MDAMiaiOECCYOXCGAVFYSPGAAILGRSirrQECLFFNANWEKDiErNQT

GVEPeYGPtDiOlRHCPATWKNISGSIEIVKQGGWLDDINCYDRTDCYEKKD

SPEVYPGGGEG^RiCNEKFSYFPEMEVrQPTSNPVTPKPPTGGGTHTCPPCPA

mXGGPSVFlXPPKPKDTLMISimXYTGVVYDVSHEGPEYKFNWYVDGYE ¥lMAETffPREEQYNSWlYYS¥lXYIXlQDWI,NGME¥EGKVSNKAIi>¥PIi

MTlSRAI£GQPEEPQVyi'LPPSglEM1®^SIT€LYlX3FYPSDrAVEWBSNG QMNYi®PYLDSDGSFFLYStePYDKSRWQ(^YFSCSVMHEALIiMi ytqRslsirpgk: csbq id mots)

This polypeptide is encoded by the following roeleic acid sequence:

ATGGATGGAATGAAGAGAGGGCrcrGGTGTGTGCTGCTGCIGTGTGGAG

CAGlQiriQGl;TTCGCCCGGCGCCGGd!AE40ITGGTAGArCAGAAA0rCAG

GAGIGTGIXTITTTAATGCTAAITGGGAAAAAGACAGAAGCAATCAAAC

TGGTGITGAACCGTGTrAIGGTGAGAAAGATAAACGGCGGGATOTTTTG

GTAGGTGGAAGAAm'nTeTGGTPeCAffTGAATAGreAAAGAAGGTTGrP

GGCrGGATGAm'IGAACTGGl'ATGAGAGGACTGAmrrGmGAAAAAAA

AGACAGCGeiGAAGIATATITClQ'rTGGlGlGAGGGGAATAlGTGIAATG

AAAAGTTTIGTTXrTTrCCOGAGATGGAAGTCAGAGAGCCCAC'ITCAAAT

CGAGTiACACGIAAGCCACCCAGGGGrGGTGGAAGXCACACArGCCCAC

CGrGCCCAGCACGTGAAGTCCTCeGGGGACCGTCAGTCTTCCTCTTCCCC

CXAAAA€CCAAfiG^CACCClCATGATCTC:€CGQACC^Gl'GA0(3'l?C^AT

GCGTGGTGGTGGACGTGAGCCACGAA0AGGCIGAGGTCAA0rrCAACl)G gtaggtggacggcgtggaggigcajaaxggcaagacaaagcgggggga

GGAOtAGTACAACAGCACGmcCGlGTGGlGAGC0rcGTCAGC<GGCtG

C^GGAGGACrGGCTGAATGOCAAGGAGTACAAGIGGAAGGt'GTCCAACA

AAaCGCTCXXmGXCCGCAjrGGAGAAAACCATCXCCAAAGCGAAAGGGCA

GGeCCGAGAAGOACAGGMAe^eXlGCCCCGATCCCGGGAGGAGArG

AGGAAGAACGAGGTCAGGClGAGCfGCCIGGIGAAAGGCrrcTATCCCA

GCGACAlGGGCGlGOAGTGG(MGAGCAAlX3GGGAGCCi3GAGAACAACT ACAAGAGeACGCClGCGGTGC!GGACTCCGACGGGXGGT?CTrxXlXTrAr

AGCAAGGi€ACCGTGGAGAAGAGCAGGrGGCAOGAGGGGAACG«lXGT

GAlGCTGCG'rGAlGCAiXMGGGffilXJCACAAGGAGXAGACGCAGAAGAG GGTCrrCCirrGTCirCGGGTAAAffiAGAATTC (SEQ K>TTO:14)

Both AeiRBa-faFc and AetRlia-mFc were remaricably amenable to recombimmi expression. As .firowa in -figure I, the protein was purified as « single, wefiilefiried peal of protein, IMbmiina! sequencing revealed a single seqiienee of™ ItGESTQE (SEQ10 NQ< 11). fiurifioafion could be achieved, by a series of eoloma Chromatography steps, inelodiag, for exarepfe, three or more of the fbltowing, la airy order: protein A elaometograpiiyi Q sepharose cinematography, phenylsepharose OlnOmatography, sigie exclaslonieBrOffiaiography, and cation exchange ehromatography* The purification could be completed with viral filtration and Buffer exchange. itbe AeiM&amp;dfire protein was purified to a purity of >9§%:as determined: by sbe exoinsioH chroinatography and >95% as determined by SOS PAGE.

ActffilaffiFe and AetEEa^nPc showed a high affinity for ligands, pafficutariy activln A. G0FG1 op

Biacore CMS chip using standard amine coupling procedure. ActR.IIa~hFe and AeiRfiarmFe proteins, were loaded onto the system, and feisding was measured, Acffilia-hFe bound to activfe with adissociation constant fEp} of 5x1 if52, and the protein boimddo GDPli wldia .¾ of 9.96x10'*, See figure 2,. AotEHarnfFe Behaved similarly.

An A-204 Reporter Gone Assay Was used to evaluate the efioets of AeiRIIa-hFc proteins o« signaling by GDF~l.l and Activin A. Coil Bus: Human Rhabdomyosarcoma ^derived.IShmsaua^d). Reportervector; pGL3{CAGA)i2 -0escribe«i:;te:jDe»n^r et.4 ϊ99$, ΕΜΒΟ 17:3Μ1-3100,) See Figure 3. The gene) , so this vector Is of general use for factors and 3..

Day 1: Split A-204 cells Into 4S~weil plate.

Day 2: A-204 ceils transfected with 10 pg pGL3(CAGA)l 2 or ρΟΑ3(€ΑΟΑ)Ι2 (10 ugH pRLCMV (! ,ug) and Fcgene.

Day 3l Add factors {diluted intoinediatnA 0,1 % BSA). inhibitors need to be pmmcnhated With Factors for 1 hr betbre adding to cells, 6 hrs later, cells rinsed with PBS, and lyse ceils. 11ns is followed by a .bneiferase assay. Typically in this assay,in the absence of any hrMhitors, AcdGn A shows roughly 1.0 ibid stinmlation ofreporter gene expression and an BDS0 ~2ttgAnL GD.P-11.: 16 fold stimulation, ED50: -1.5 ug/ral. GDF-S shows an effect similar to. GDF-11,

As shown in figure i; AelRlIaAFc and ActKIiannFe mhibli GDF-8 hiediated signaling at pieontoiar concentrations. As shown in figure,5, three

AcfRIXa-hFo inhibited GDF4d· aalGSO of approximately 200 pM,

The ActRIIa-BFc was very stable in pharntaobhinefic studies. Rats were: dosed with 1 mg/kg, 3 mg/kgpr 10 mg/kg of ActRilfehFe protein and plasma levels offhe potein were measured at 24, 48, 72,.144 and 168 horns, In a separate study, rats were dosed at 1 mg/kg, 10 aig/kg or 30 mgAg, In rats, AetRiia-Mte had an 11 -14 day serum half life and circulating levefe of the drug were guite-htgh after two weeks (11 pg/ud, i admluistrations of I mg/kg, 10 mg/kg or 30 mg/kg, respectively.) In cynomoigus monkeys, the plasma half life was substantially greater than. Iddays and circulating levels of the drug were 25 pg/mi, 304 .pg/mi or 1440 pg/mlidr initial adm hrisiraclons of Tmfykg, 10 mg/kg or 30 mg/kg, respectively. Pfeiknioarv results In humans suggests that the serum half life is between about 20 and 30 days.

Example 2: Aotlflla^mim PronmtesBene Growth In Vivo

Normal fertile mice (B^LB/β): were d5spd with AotRIfesEe ah a level oft mg/kg/dose, 3 tngAsg/dose or 10 :Big/kg/desegw:itfc doses gives twice weekly- Bose misers! density arsd bone mineral content were determined by 'DEAA, seeyfigure 6. 1« ΒΑΪ^Β/e female mice, fiBXA scans: showed a- sigsnfieantinc3»ase ^20%) in bone mineral density and content as a reanlt of AetfUIa-niEe treatment. See figures17 and 8:,

Tbns? antagonism of ActEHa caused increased bone density mid eonterd in iprmaliemale nilce. As: a next steps the ef&amp;et of AciRHa-mFo an bone In a monss modelfor oAeo|?orostS: was tested,

Anderssemet aL ¢2061), established that ovariectomiaed mice suffered substantial bone i0ssy{;rongly 50% loss of trabecular bone six; weeks posbopedulen), and dial bone loss in these mice could be edrreeted: wltfe candidate tberapedile agents, snob as parathyroid hormone,

Applieaats tped C57BLd female mieefhai: were ovariectonrkted fOV3£> or simm operated at 4-5 weeks; of age, Eight weeks after surgery, treatment wih AetMiaHAFb (10 mg/kg^ twice we#ly) or coptrol fFBSf was initiated. Bone density was measured by CT scanner.

As shown in figure 9S loss of trabecular bone density relative to the sham controls after six weeks.. Ast^Ia-mFdi^tm^.fs^mdtbon© .of&amp;e sham operated mice.

At 6 and 12 weeks of fe treatment AcfRifemtFe cansed substantial merease in trabecular bone of OVX mice* See figure 10, hone density increased fey;.2A% relative .to .P&amp;$;:ca®im!s, After 12 weeks, the increase was 27%. in the sham opiated mice, AetRfiaemFc also caused a shbstantM Increase In trabecular bone.. See figure if. After 6 and 12 weeksfthe treatment produced a 35% increase relative to controls. ίο aiv additional set of espstanoats, ovariectomhsed (0¥%) pr sham operated mice as described above warn treated witbAetRitenFe (10 mg/fcgf twice weekly or control ( PBS) over twelve weeks. Similar to the results described above tor AetRlIa~mPc, OVX mice receiving AclRila-mFc exhibited: an increase in trabecular bonedepsby of 15% by as early as.foer weeks and 25% alter 12 weeks of treatment -12).-. Sham operated mice receiving AetEilamrPc similarly showed an increase in trabecular bone density of 22% by as early as four weeks and of 32% ate 12 weeks of treatment-(Figure13).

Ate twelve weeks of treatment with AetRla-mFe, whole body and ex vivo feihcr 0Β.ΧΑ analysis showed that indnees an inctese in bone density in both ovariectomlaed and sham operated mice (Figures 14A and MB. respectively). These results are also supported by er vivo pQCT analysis of the femoral nndshaft which demonstrated a significant increase in both total and cortical bone density ate twelve weeks pftms^^twilfi..Atd^3^mF0v ¥ehiele^neated control oyarieeiomixed mice exhibited hone densities that were comparable to vehfeie··· 'treated control sham operated mice (Figure Ϊ 5). In addition, to hone density, bone conteiF increased fpllowing AeffilamiFC treatment Ex vim pQCT analysis of the femoral aridsbaft demanstrated a significant increase: in both total aud cortical bone contentate twelve weeks: of treatment widt AetRIlamiFc while bottj ovarlectommed: and sham operated vehicle eomrol-treated mice exhibited comparable: bone content: (Figure Id). Ex vivo pQCT analysis of the femoral midshaft: also: showed that AetRXia~mf c treated mice did not show a change in periosteal circumference·; however AmBIIa-mFc treatment resulted In a decrease in endosteal cirenmlersnee indicating aa increase in. cortical thickness due to growth: on the inner surface of the femur (Figure 17),

Mechanical testing of femurs determined that ActREamiFe was able to increase the extrinsic characteristics of thefeone (maximal load, stiffness- and energy to break) which contributed to: a significant increase in the intrinsic properties (ultimate strength) of the bones. Ovarieetomized mice treated, with AeiSJIa-mPc exhibited increased bon® strength to levels beyond sham operated, vehidie treated controls, indicating a eourpiete mversal of the osteop^jde.-jteiotyp(B: CRigBfeTS). increase bone. density In normal female mice and, fertkermore, correct defects in bone density, bone content. and «Mmafeiy bene strength. in a mouse model of osteoporosis. .la a:fbrfeer.s&amp;of ex^eife^afe»#ke wme ov&amp;rieetomfeed or shani operated at 4 weeks, and beginning at 12 weeks received either placebo or ^pt^ia^Fc (2 times/Week, Ifeigdrg) (also referred teas ilAP-11 k Figotes 1^-24), a ferfer period of 12 weeks, A variety of bone parameters were evaluated, As slmwn its Figure 19, AefMIamiFc mereased wtebral trabecularbone Volume te total volume ratios (BV/Tf) in both the D¥X aad SHAM operated: mice. AetSdiamilfe also improved the trabecular arebfectare (Figure 20% increased coAfeslfeicimess (Figure 21) and improved bone strength (Figure 22). As shown in Figure 23, ActRIIa-mFc produced desirable effects at a range of doses irons fmg/kg to id mg&amp;g.

Bode Mstomorphometry was conducted at a 2 week time point in sham operated mice. These data* presented in Figure 24- demonstrate that AeiRI&amp;mrFc has a dual effect, both inhibiting^bene resorption and promoting bone growth. Thus AciRFiaouFc stimulates bone growth (anabolic effect) and inhibits bone resorption (anri-caiabolie effect).

Example 4: Alternative ActRHa-Fc Proteins

An: alternative construct may have a deletion of tbe CAerminal 1. 5 amino abide of the eklraceltuiar domain of AetRIia. The sequence for such a eorssimct Is: pmsernedbbldw (Fc portion under|ined)(SEQ If) NO: 12); lLGfeSETtp3iiTi%NWEiC0^

ErVlSQOGWEpWNETORTDCVEIGfDSPEVYFCCC^ONMCNEKFSTFFEej; GG'PHTEiFP€BAPEELG:OFS\hLFPPKPKlP'lXhtlNR’rFE¥TT2\’4ATtV'SBEDPE¥, KPHWYf DGVBYHHAErKFREBO YNSTYRV VY v LTYLKODW LNGKEYRCK VSNKAWFiBRTlSRAfeGfipRFi^TVYTLPPSREbhrrKNDVSLTCLVICGPYPS Dl A VE WESHGOPENHYKTTFPYLDSPGSFFI-YSKif i V DKBRWOOOhiYPSGS VMHEALirfNHYTaKSfAESPefe

BY REFERENCE

AH publications and msHRoneH reference in tE eir as ifeach individual ivubiicatioii or patent was specitleaUy and Individually Indicated to be Incorporated:by referencfe: WHile specific embedments of the subject matter Have been, dismissed, ibe above specification is iiiustraike and not restrictive. Many variations will become apparent to those skilled in. the art upon review of this specification and the claims below. The tUO scope of the invention, should be determined by reference to the: claims, along with: their Ml scops ofeqnivaietas, and. the speeiBeation, along with such vaxiatious.

Claims (41)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:-

   1. A polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 3, wherein the N-terminus of the polypeptide is ILGRSETQE.
2. 2. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 3.
3. 3. The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 3.
4. 4. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 2.
5. 5. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 2.
6. 6. The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2.
7. 7. The polypeptide of any one of claims 1-6, wherein the polypeptide is a fusion protein that further comprises one or more heterologous polypeptide domains.
8. 8. The polypeptide of claim 7, wherein the polypeptide comprises a heterologous polypeptide domain selected from an immunoglobulin Fc domain or a serum albumin domain.
9. 9. The polypeptide of any one of claims 1-8, wherein the polypeptide comprises one or more modified amino acid residues selected from: a glycosylated amino acid, a PEGylated amino acid, a famesylated amino acid, an acetylated amino acid, a biotinylated amino, an amino acid conjugated to a lipid moiety, and an amino acid conjugated to an organic derivatizing agent.
10. 10. The polypeptide of any one of claims 1-9, wherein the polypeptide is glycosylated and has a glycosylation pattern obtainable by expression in a CHO cell.
11. 11. The polypeptide of any one of claims 1-10, wherein the polypeptide binds to activin.
12. 12. The polypeptide of claim 11, wherein the polypeptide binds to activin A.
13. 13. The polypeptide of claim 11 or claim 12, wherein the polypeptide binds to activin B.
14. 14. The polypeptide of any one of claims 1-13, wherein the polypeptide binds to GDF11.
15. 15. A pharmaceutical preparation comprising the polypeptide of any one of claims 1-14 and at least one pharmaceutically acceptable carrier.
16. 16. The pharmaceutical preparation of claim 15, wherein the preparation is pyrogen-free.
17. 17. An expression vector comprising a polynucleotide comprising a coding sequence for the polypeptide of any one of claims 1-14.
18. 18. A cell comprising the vector of claim 17.
19. 19. The cell of claim 18, wherein the cell is a mammalian cell.
20. 20. A method for promoting bone growth, increasing bone density, increasing bone strength, treating or preventing a bone-related disorder, promoting bone growth and inhibiting bone resorption, inducing cartilage formation, increasing bone mineralization, preventing demineralization of bone, treating a bone fracture, or treating a cartilage defect in a subject, the method comprising administering to a patient in need thereof an effective amount of a polypeptide of any one of claims 1 to 14.
21. 21. The method of claim 20, wherein the subject has cancer associated with bone loss.
22. 22. The method of claim 20 or claim 21, wherein the subject is the recipient of a cancer treatment regimen that is associated with bone loss.
23. 23. The method of any one of claims 20-22, wherein the method is for promoting bone growth in a subject in need thereof.
24. 24. The method of any one of claims 20-22, wherein the method is for increasing bone density in a subject in need thereof.
25. 25. The method of any one of claims 20-22, wherein the method is for increasing bone strength in a subject in need thereof.
26. 26. The method of any one of claims 20-22, wherein the method is for promoting bone growth and inhibiting bone resorption in a subject in need thereof.
27. 27. The method of any one of claims 20-22, wherein the method is for inducing cartilage formation in a subject in need thereof.
28. 28. The method of any one of claims 20-22, wherein the method is for increasing bone mineralization in a subject in need thereof.
29. 29. The method of any one of claims 20-22, wherein the method is for preventing demineralization of bone in a subject in need thereof.
30. 30. The method of any one of claims 20-22, wherein the method is for treating a bone fracture in a subject in need thereof.
31. 31. The method of any one of claims 20-22, wherein the method is for treating a cartilage defect in a subject in need thereof.
32. 32. The method of any one of claims 20-22, wherein the method is for treating or preventing a bone-related disorder in a subject in need thereof.
33. 33. The method of claim 32, wherein the disorder is associated with low bone density or strength.
34. 34. The method of claim 32 or claim 33, wherein the disorder is selected from the group consisting of: primary osteoporosis, secondary osteoporosis, post-menopausal osteoporosis, hypogonadal bone loss, tumor-induced bone loss, cancer therapy-induced bone loss, bony metastases, multiple myeloma, Paget’s disease, hyperparathyroidism, Cushing’s disease, renal tubular acidosis, and kidney disease.
35. 35. The method of claim 34 wherein the disorder is multiple myeloma.
36. 36. A method of making an ActRIIa polypeptide, comprising: a) culturing a cell under conditions suitable for expression of the ActRIIa polypeptide, wherein the cell comprises the vector of claim 17; b) recovering the ActRIIa polypeptide so expressed. 37 The method of claim 36, wherein the cell is a mammalian cell.
37. 38. The method of claim 37, wherein the cell is a Chinese hamster ovary cell.
38. 39. The method of any one of claims 36-38, wherein the polypeptide is expressed using a tissue plasminogen activator (TPA) leader sequence.
39. 40. The method of claim 39, wherein the TPA leader sequence comprises the amino acid sequence of SEQ ID NO: 9.
40. 41. An ActRIIa polypeptide when produced by the method of any one of claims 36-40.
41. 42. Use of a polypeptide of any one of claims 1 to 14 for the manufacture of a medicament for promoting bone growth, increasing bone density, increasing bone strength, treating or preventing a bone-related disorder, promoting bone growth and inhibiting bone resorption, inducing cartilage formation, increasing bone mineralization, preventing demineralization of bone, treating a bone fracture, or treating a cartilage defect.