AU2015202418B2 - Designer osteogenic proteins - Google Patents

Abstract

Abstract of the Disclosure The invention relates to novel designer osteogenic proteins having altered affinity for a cognate receptor, nucleic acids encoding the same, and methods of use therefore. More preferably, the novel designer osteogenic proteins are designer BMPs and have altered affinity for a cognate BMP receptor. The designer BMPs demonstrate altered biological characteristics and provide potential useful novel therapeutics.

Description

DESIGNER OSTEOGENIC PROTEINS

The present application is a divisional application of Australian Application No. 2011292810, which is incorporated in its entirety herein by reference.

FIELD OF THE INVENTION

This application relates to the field of osteogenic proteins, methods of making improved osteogenic proteins, and methods of treating patients with osteogenic proteins.

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.

The cystine knot cytokine superfamily is divided into subfamilies, which include, the transforming growth factor β (TGFp) proteins, the glycoprotein hormones, the platelet-derived growth factor-like (PDGF-like) proteins, nerve growth factors (NGF), and the differential screening-selected gene aberrative in neuroblastoma (DAN) family (e.g., cerberus). In turn, the ΤΰΡβ superfamily comprises approximately 43 members, subdivided into three subfamilies: the ΤΰΡβε, the activins and the bone morphogenetic/growth differentiation factor proteins (BMP/GDF).

The TGF-β superfamily members contain the canonical cystine knot topology. That is, cystine knots are the result of an unusual arrangement of six cysteine residues. The knot consists of bonds between cysteines 1-4, cysteines 2-5, and the intervening sequence forming a ring, through which the disulfide bond between cysteines 3-6 passes. The active forms of these proteins are homodimers or heterodimers. In each case the monomer topology is stabilized by the cysteine knot and additional cysteines contribute to additional intrachain bonds and/or mediate dimerization with another protein unit. See Kingsley, 1994, Genes Dev. 8:133-146; Lander et al, 2001, Nature 409:860-921. BMP/GDFs are the most numerous members of the TGF-β protein superfamily. The BMP/GDF subfamily includes, but is not limited to, BMP2, BMP3 (osteogenin), BMP3b (GDF-10), BMP4 (BMP2b), BMP5, BMP6, BMP7 (osteogenic protein-1 or OP1), BMP8 (OP2), BMP8B (OP3), BMP9 (GDF2), BMP10, BMP11 (GDF11), BMP12 (GDF7), BMP13 (GDF6, CDMP2), BMP15 (GDF9), BMP16, GDF1, GDF3, GDF5 (CDMP1; MP52), and GDF8 (myostatin). BMPs are sometimes referred to as Osteogenic Protein (OPs), Growth Differentiation Factors (GDFs), or Cartilage-Derived Morphogenetic Proteins (CDMPs). BMPs are also present in other animal species. Furthermore, there is some allelic variation in BMP sequences among different members of the human population. BMPs are naturally expressed as pro-proteins comprising a long pro-domain, one or more cleavage sites, and a mature domain. This pro-protein is then processed by the cellular machinery to yield a dimeric mature BMP molecule. The pro-domain is believed to aid in the correct folding and processing of BMPs. Furthermore, in some but not all BMPs, the pro-domain may noncovalently bind the mature domain and may act as a chaperone, as well as an inhibitor (e.g., Thies et al., Growth Factors 18:251-9(2001)). BMP signal transduction is initiated when a BMP dimer binds two type I and two type II serine/threonine kinase receptors. Type I receptors include, but are not limited to, ALK-1 (Activin receptor-Like Kinase 1), ALK-2 (also called ActRla or ActRI), ALK-3 (also called BMPRIa), and ALK-6

(also called BMPRIb). Type II receptors include, but are not limited to, ActRlla (also called ActRII), ActRllb, and BMPRII. The human genome contains 12 members of the receptor serine/threonine kinase family, including 7 type I and 5 type II receptors, all of which are involved in TGF-β signaling (Manning et al., Science 298:1912-34 (2002)), the disclosures of which are hereby incorporated by reference). Thus, there are 12 receptors and 43 superfamily members, suggesting that at least some TGF-β superfamily members bind the same receptor(s). Following BMP binding, the type II receptors phosphorylate the type I receptors, the type I receptors phosphorylate members of the Smad family of transcription factors, and the Smads translocate to the nucleus and activate the expression of a number of genes. BMPs are among the most numerous members of TGF-β superfamily, and control a diverse set of cellular and developmental processes, such as embryonic pattern formation and tissue specification as well as promoting wound healing and repair processes in adult tissues. BMPs were initially isolated by their ability to induce bone and cartilage formation. BMP signaling is inducible upon bone fracture and related tissue injury, leading to bone regeneration and repair. BMP molecules which have altered affinity for their receptors would have improved biological activity relative to the native proteins. Such BMPs include proteins with increased in vivo activity and may provide potential improved therapeutics for, among other things, tissue regeneration, repair, and the like, by providing greater or altered activity at lower protein levels thereby providing improved protein therapeutics.

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.

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”.

SUMMARY OF THE INVENTION

The invention includes a designer BMP protein comprising at least one mutation in at least one type I or type II receptor binding domain, wherein the mutation confers altered binding to the type I or type II BMP receptor compared with the binding to the type I or type II receptor by a corresponding wild type BMP.

In a first aspect of the invention there is provided a designer BMP protein comprising: a first set of mutations with respect to SEQ. ID NO:1 comprising V33I, P36R, H39A, H44D, P48S, A52N, D53A, L55M, S57A, N68H, S69L, V70M, insertion of a P after N71, S72E, K73Y, I74V, A77P, and V80A, wherein the designer BMP protein exhibits altered binding to each of a type I and a type II BMP receptor compared with the binding to the type I or type II receptor by a corresponding wild-type BMP.

In a second aspect of the invention there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding the designer BMP protein of the invention.

In a third aspect of the invention there is provided a designer BMP protein comprising the following mutations with respect to SEQ. ID NO:1: V33I, P36R, H39A, H44D, P48S, A52N, D53A, L55M, S57A, N68H, S69L, V70M, insertion of a P after N71, S72E, K73Y, I74V, A77P, V80A, E83K, S85R, A86P, I87M, L92Y, E94D, N95G, E96Q, K97N, V98I, V99I, L100K, N102D, Y103I, D105N, V107I, G110E, and R114S, wherein the designer BMP protein exhibits altered binding to each of a type I and a type II BMP receptor compared with the binding to the type I or type II receptor by a corresponding wild-type BMP.

In a fourth aspect of the invention there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding the designer BMP protein of the invention.

In a fifth aspect of the invention there is provided a designer BMP protein comprising the following mutations with respect to SEQ. ID NO:1: V33I, P36R, H39A, H44D, P48S, A52N, D53A, L55M, S57A, N68H, S69L, V70M, insertion of a P after N71, S72E, K73Y, I74V, A77P, V80A, E83K, S85R, A86P, I87M, L92Y, E94D, N95G, E96Q, K97N, V98I, V99I, L100K, N102D, Y103I, D105N, V107I, G110E, and R114S, having an affinity for the ActRllb receptor of less than 1 nM.

In a sixth aspect of the invention there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding the designer BMP protein of the invention.

In a seventh aspect of the invention there is provided a designer BMP protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NQ:70.

In an eighth aspect of the invention there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding a designer BMP protein of the invention.

In a ninth aspect of the invention there is provided a method of producing the designer BMP protein of the invention comprising introducing a nucleic acid encoding the designer BMP protein into a host cell, culturing the cell under conditions where the protein is produced, and purifying the protein.

In a tenth aspect of the invention there is provided a designer BMP protein when produced by the method of the invention.

In another aspect, the protein is selected from the group consisting of BMP2, BMP4, BMP5, BMP6, BMP7, BMP8 and BMP9.

In another aspect, the protein comprises at least one mutation within: the type II binding domain A; the type II binding domain B; the type I binding domain; and any combination thereof.

The invention also includes a designer osteogenic protein comprising an amino acid sequence comprising at least one mutation in at least one type I or type II receptor binding domain, wherein the mutation confers altered binding to the type I or type II BMP receptor compared with the binding to the type I or type II receptor by wild type BMP2.

In one aspect, the mutation is a mutation within the type II binding domain A wherein said mutation is at least one mutation selected from the group consisting of a mutation at V33, P36, H39, and F41 with respect to the sequence of SEQ ID NO:1.

In another aspect, the is a mutation within the type II binding domain A wherein said mutation is at least one mutation selected from the group consisting of V33I, P36K, P36R, H39A, and F41N with respect to SEQ ID NO:1.

lo ye? another aspect the mutation *s a mutation wlMmiAe type It binding domain B whenem SPid: mutation is at least one mutation selected from the group consisting of a mutation at E83, S8f>, M89: 693, E94, E36. K97, and V99 with respect to the sequence ot SAG ID NO: 1

In a iunher aspect, the mutation is a mutation within the type It binding domain B wherein said mutat.son 1¾ at least one mutation selected tram the proop consisting of E83K, 8S5N. M89V. LOOF E84Q,, £968. K97N. and V93I with respect to of SEG tD NO:1. in another aspect, the mutation is a mutation within the type i binding domain wherein s&amp;fcfr mutaton is at least one mutation selected from the group consisting of a mutation at H44. P48, AS2< 0¾ 1.S5, SB?, N68, 889, V70, an insertion of a single amino acid after N71, S72, K73. 174, A??, and y'SO with respect to trie sequence of SEG ID NO: 1. in yet another aspect, the mutation A; a mutation within the type I binding domain wherein sain mutation is at least one mutation selected from the group consisting of H44D. P48S. A52N* 0§3A: Labfd S5?A; NS8H. S»9l.. V70M. insertion of P after N?l 8726, K73Y 174ν', A77P and V80A With respect to the sequence «?' SEQ ID NO 6 in a further aspect, trie protein comprises a mutation at each of amino adds Η44; F48, AS2, DBA: EBB. 857, N88. 869, V?0. insertion pf a single amino acid after N7i, 872.. K?3, 174,, A77, and Y80- With respect, ίο the sequence of SEQ ID 740:1,

In anothar aspect, the ptofe-n composes a muiatbn at each of amino acids H«4. P48. An? Dp3 LSS, S57. Nod, S>59, V70, insertion of a a;; Kite amino acid after N7'6 S72 K73, I At, A??, and V80 with respect to the sequence of SEQ ID NO' 1 wherein the mutations are H44D, P48S. AB2N. D53A, LbSEI, SB?A. N6SH, S63L, WOM. insertion of a P after N? i. S?2E, K73Y. i?4V. At 7P, and V80A. in yet another aspect, the protein comprises a mutation at each of amino acids V3-3> P3S, H3S; Site. f\4B9: LS2, 694. 696. K97. arid V99 with respect to the sequence of SEQ ID NO.1

in another· aspect the protein comprises a muradon at each of ammo acids V33. P3G. H39, SS5. 1839. L92; 694 696: K97, and VS9 with respect to the sequence of SEQ ID NO.1. wherein the mutations are V33I. P3SK. H39A S8SN. M89. L92F. E94D, 6368. K97N and V99I in a further aspect, the protein comprises a mutation at each of amino aceis V33, P36. H39, i-M4. P43, A62, DBA l.£5. S57. N6S, SG9, V?Q< insertion of a singie amino acid after N?1. S72. K?3. t?4. A77. and V80. S8S, 6949. 192, ESA E96. K97, and V99 with respect to the sequence of SEQ ID NOi

In yet another aspect, the protein comprise? a mutation at each ot amino adds V33: PuA. H39, H44, P48. A.32. 063. 636, S37, N-38, S-39 V70. irisertion of a singie ammo add after N71.S72 K73. i74. A??, end VSO. 886, M8Q, t.92, ES4. ESb, K9?; and V99 with respect to the sequence of SEQ ID NO:1 wherein the mutations are V336 P38K. H3S.A. 1-1440. E43S, A62N, D33A. L56M, S67A, N-SSH. 61696. V70M. insertion of a P after N?f. 8726. K?3Y. 174 V, A??P. and VSOA, SSSN, 6169, 692F, 6940, 6968 K97N. arm V99I.

In yet another aspect, the protein composes a mutation at each of amino adds V33. 936. H39. Had, F^6, A.52. D63, 1.58 SS7. N68, SCiS. V'7D, insertion of a single amino acid after Ν7ϊ. S72, K73, 174. 192, £94, E96, KteAand V99 with respect to tee sequence ofSEO ID N0:1 V33I, P36R. H39A, H44ft· E4S£h Ao2M, PSSA, LS5MI S57A, N8SH; SGSL WOM. insertion of a P after N71, S72E K73Y. 174V, A77P. and VS9A, S85N, Km, I S2F. F94D E&amp;8S. KS7N, and V99I. inanother aspect. the protein bind.-;: the A1 K.2 receptor vote a K;> not greater than aboutS' nM, the Α£Κ3 receptor with a K<> not greater than about 2 nM; teo AlKS recaptor w*ih a K ; not greater than about ! nM: ids AetRMA receptor web a kA not greater than about ? nM; the AoiRhB receptor with a K:> not greater tears aUoul 0,5 nM. and the UMRRIiA receptor with a K.-, not greater tears about 3 5 nM, in one aspect the protein further comprises ?, 2, 3 4 5, 6, 7, 8 3 or ID amino aod roatpiippa not located withm if m type t or the typer ti tending regions.

The invention includes a designer osteogenic protein eompnsshg tee amino acid ssqpegha &amp; any one of SEG sD NOs.8-73

The invention includes a designerrosteogepic proteinjcompsing the amino acid .sequence of SEO ID NO-12

The invention inotedea ai gessgnar osteogenic: propp Opfhprislng tee amlng acid leqtfenGe of S£Q ID NO, 14,

The itevemion tnoindes a designer obtedgehic: protein :ete*phsihg tee asrslnd. aoid sequence of: SEO ID NOG6

The invention includes a Jeetgner osteogenic protein eempfiairsg tee erotho acid sdgitersbe fd; SEO ID NO:37

Th§;. ..invention: rhda#a a detegner BMP protein eompnalig at leapt one mutation in at least one type .! or type it receptor birsdis-ig domain, wherein the mutation confers altered binding to tee type I or type H the binding to tee type if receptor by a corresponding Wild type BMP The soothed comprises introducing a nucleic acid encoding the protein into a host ceil, culturing the Pelf under conditions where the protein is produced, and purifying tee proteirh in one aspect, tee nucleic acid composes » sequence selected hose tee nucleic acid sequence of any one of SEG iO NOs :74-139.

Tite snyentton includes a designer Mite protein composing an amine acid sequence «temphstiig : at ieasi one mutatten in at least one type d or type it receptor binding; domain, wherein ted muiatiph oontete pifered binding to the type f or type if '8|$* t0<tept«r g«^^^Lv4!iji'''the binding te the; type 1 or type i| receptor by wild type BMPS. in one aspect, tee mutation is a mnuifon within the type ti binding domwn A wherein said mutetson is si toast one mutation selected from tee -group consisting of a mutation si 157, K6D, G61 A53 N6G V66, and Odd wim respect to the sequence of AEG :D NO:4.

in artothsr aspect, the imitation is a mutation within the type !i binding domain B wherein said : mutation is at least one mutation selected from the group consisting of KIDS, Ml 10. A111. V114, F117, D1 F7 Nt?0 St/Ί, N1??. v 5?3. anl: 134 wsto νπνο* to Ά* sequence of SEQ A- NO A

In yet another aspect, the muiat:on is a mutation within the type I binding domain wherein said mutation is at least one mutation selected from the group consisting of a mutation at S?2, N76. A77, H7S, ms. NSG, A81: NKk VS?. 139. H92, L93, MM. N95. P9€. £§?, ¥90,3/99, ahd PlOO with respect to the sequence of SEQ tD NO:4. in another aspadt, ins mutation is a-mutate'at oaah of amiho acid residues IS?, KSQ, Q01f Ate N68. ¥86, and 068 with respect So the sequence of SEQIQ NQ;4,

in a further aspect, the mutation is a mutation at each of amine acid residues KIDS; M110, All 1,. Vt 14« F117, VI as. and 1124 vs#t resect to toe amino odd seqteda Of SEQ !D NO:4. :n yet another aspect, the mutation is a mutate at each of amino acid residue^ S72. '476. A?7, H70 M79 N80, A8:. N83, V87, T89. H82. 193, M94, N9b. P96, E97, Y98, V99, orE'ffXl with msnechto the amino acid sequence of SEQ SD NCH, ;n another aspect the designer 8MP8 protem somptfsing an amino Add sequence comprising at least one mutation in at least one type I or type ii receptor binding domam,: Wherein the mutation confers ohe- ed binding to the type : or type ii BMP receptor compared with the binding to the type I or lype U receptor by wsid type BMP6 turther comprises 1, 2, 3, 4, 5, u, 7, 8. 9, or 10 amino acid mutsttforrs not located within the type I or me type ii binding domains.,

The invention: indudes an isolated nucleic acid mdfeouie eompdsihg a nucleotide sequence encoding an amino arid sequence selected from the group comdsfing of the sequence of SEQ ID NOs'8 to 73. in one aspect., the nucleic add encodes a protoin comprising an amino add sequence selected from the group consisting of the sequence of SEQ ID NQ:12, SEQ ID NO;14, BlQ ID NQfteahd SEQ: ID 140:37.$$

The invention includes an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs 74 to 139. in one .aspect, the mieieip -acid sgmpnses a nticieufide segiience seieded fmm ftto group"· consisting of the sequence of SEQ ID NG:78, SEQ tD NQ:8Q. SEQ »D NO: 1¾ and SEQ iDfT0;103.

The invention inciudes p mefttod of producing the designer 8ΜΒ0 protein oompndng an amino acid sequence comprising at least one mutation in at least one type I or type ii receptor binding domain. #ieteirs the putotion confers altered, binding to toe type I «Y type::1i BMP rertoptor oorapared with the binding to the typo t or type it receptohby wild type BPP8. The method Comprises mtfodudng a nuoldo acid encoding said protein into a host ceil, cuiluring said ceil utvier conditions where said protein is produced, and purifying said protoin.

The invention includes a method of treat: n§ a bone disease associated wife bone loss in a pedant in need teemfe, The method comprises administering a therapeutically effective amount of a designer BMP protein oompiisteg at least one mutation in at feast one type i or type H receptor binding domain; whesem the mutation confers altered binding to the iypa i or type ii BMP receptor compered rath the binding to the tvpe i or type II receptor by a l?3' treating bone disease in tee patient

The invention includes a method -of treating iSbmsis te a paheot jit need thereof· The method comprises administering a teerapeuticaiiy affective amount of P designer BMP protein fidrppbing ^ feast one mutation in at least one type i or type II receptor binding domain, wherein the mutation confers altered binding to the type I or type: With the binding to the type f Of type it receptor by a corresponding wild type BMP to the paipni, thereby treating fibrosis.

The invention inciudesi aifeethod of inducing. bogs formation m a tissue. The method comprises contacting the tissue with a designer BMP protein: comprising at feast one mutation In at feast one type Γ or type li receptor binding domain, wherein the mutation oopferp altered binding td the type I or type hi. BMP receptor compared with the binding to the type I or type ii receptor by a corresponding wild type BMP:;thereby inducing bone formation ό sate tissue

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of idstrsting tee invention, teens are dagiet&amp;d'' .jd^^gor-ogrtgih' embodiments of the invention However, the invention Is not limited to the precise arrangements add instfelrriontelfifos of itie embisdimenfs depicted m the drawings.

Figure t, comprising panels A-G, is, a diagram showing the alignment of various wild type and designer BMP ammo acid sequences and indicating (by being within a box) the regions dFfeese proteins potentially involved In type I and type ii: receptor interactions:. Figure 1A shows the amino acid sequence; alignment of wild type BMP 2, 8MP4, BMPofeSMPB, BMP?, 8MP8 bhd 8MP9, ; Pigufe IB shows tee amino acid sequence alignment of various designer BMPs where the corresponding wild type BMP is BMP2. Figure 1C shows the amino odd sequence alignment of various designer 8MP8 molecules where tea corresponding wild type BMP is 8MP6.

Figure 2 is ao lfustratioh of ai struefptef modet showing » wild type SMP2 homodimsf binding to two type i and two type ii BMP receptors.

Figure 2 composing nanels A and 8 w an timgMm of a structural model showing the position of the histidine doorstop iHS4) in human BMP2 produced in Chinese Hamster Ovary (CHD) (Figure SAI and F. col; ceils frigate 3Bs I icurr 4 oompuscw van sis A one 8 s s esagfe n r u He ling the location o! the giycan tether arid potential histidme (His) doorstop. Figure 4A shows the giycan tether -id-linked giycan at Hod} and histidine 54, in the nou-dooistep onenhfeon as well as fee interaction of the giycan tether with P1S ais in CHO-produeed BMP? Figure 4B shows -he giycan fethei (M-iinked giycan at NBO) and tee histidine in mn-dw ^op oonfiQvki sur at h?f (r hv?1" as wG a- "he ^ ecnespo ?< n g ίο -ί 1 -¾ m -AdC' } h<* s^ep^ aiigpffigpi^ of 8MP2 (11-KSSCKRHP) and BMP&amp; (3S-KTACRKH^ showing the corresponding amino acids between BMP2 and BMP6 is shown along She top of the figure.

FighteS* composing panels A-D* stiows pf ' desioner BMPs. Figure QA shows a chromatogram showing gradient elution of BMPs using a ceiiuiihe suiiate column. Figure SB so an image of a Coomass-e stained SDS-PAGE (non· reduced on the feff and reduced on the right side; gas containing samples of fractions from the oeiiufine sulfate column step f igure 5C shows &amp; chromatogram showing the profile from preparative reversed phase purification step Figure 5D Is an image of a Coomeosse stained BOS~PAGF (non-reduced on the Aft and reduced on the right) gei of BMP containing samples of the tractions obtained Oy the preparative reversed phase purification step.

Figure δ, comprising panels A-0, show images of Coomassietorfained SDS-PAGF protein gels showing pudfiad BMP2 wild type and various mutants as Indicated along the lop of eachgel Image.: The gels wvbro run under either nofpfsducing (Fipres SA and 9¾ and; reduefag IRgutes BO arid PD) conditions.

Figure T stews alkaline phosphatase assay results ir» C2C12 pte-myoblasts cumpahag the osteogenic activity of wild type BMP2 and 8MP2fi| heterodimsr to fie various designer BSyPd as indicated ;n the graph legend •Figure B shows the results of a .02.0:2' BMP'RespOhsO Bdimeht tucifFfase (©BBridciMrase) assay indicative of Smad activity showing stronger signing by SMPi oontpomd to BMP! and sgafyafesnf signaling to 8ΜΡ2ίβ..:

Figum u comprising panels A and B shows the ectopic hone fermafton mediated by Sanous BMPs. Figure 9A is a graph showing the amount of ectopic bom; icaicuisted- as mitgramS # hydroxyapatite; mg HA) as determined by uCT analysis fori fmfteed with the indicated BMP (BMP2. BMFE, and BMP2'fF at the dose indicated (D.1 or 0.5 pg). Figure B8 is a graph showing the amount of eefopto hone (raicuiafed as m^rarofFff By μΒΤ analysis for each limb which was iropianlad with the indicated BpF {SMP2, SMPC3, pMFA. and jBfetFF) at the dose indicated (BJ or 0.5 pg). The data presented are front 2 separate experiments. f igure 10, comprising panels ΑΌ. shows images of radiographs showing the results of a non-human primate tN HP) fibula osteotomy model at 4 and S weeks. Radiographs are shown of she fibulas of 7 representative NHPs that received BMPE and BMPG, respectively. at 0.S mg/ml (250 «g total BMP daiivered/llmb), Faeh NHP received WT BMF3 at fhs same dosa in the pontralaferai Slmh. Figures 10A and 10B show the radiographs tor the NHPs indicated at the top of each diagram showing the effects M 8MPF; compared with BMP2 w?ld type At 4 weeks and δ weeks, respestiyely. Figures ICC and 10D show the radiographs for the NHPs iridlcafed at «to top of each diagram showing the effects of BMPG compared with 8MP2 wild type at 4 weeks <md B weeks, respectively.

Figure 11 is a graph shmwibSMlhSf -i&amp;ene- vv5SrfM«T|0- rgf ihl isftfNlimited With B|4f5-€i versd* xespiaiateral limbs treated with SMP-2y.

Figure 12 h, a graph showing ϊ smelts af an alkaline phosphatase assay m C2C"? pre-myobissts composing the osteogen-c activity of wild type BMP2 end BMP-GBR, BMP-GuP. and BMP2'6 heterodimer.

Figure 13 is a graph showing the amount d ectopic bune {edeuiatdd as milligram;. oi hydrei>ryapatite) as determined by gCT analysis ter each «mb wdch was implanted with the indicated, BMP {BMP-2, 8ΜΡ-2ιδ, SMP-E, BMP-GLR, and BMP-6} at the dose iOdteated iO 0$; hr 0,26μ§).

Figure 14 is a graph showing the amount of ec-opte hope icaicuiated as miliigrapia jsf hydroxyapatite) as determined by pCI anaiysrs for ssch limb Which was Implahted' taifih Ihfc''fct0i«|&amp;6 BMP {BMP-2, 8MP-2<6. BMP-E, BMP-GER. and 8MP-6) at the docs indicted #.0B or 0J5M0;pThesa are the results from an e^asnmdnt separate from that shown "in-Figure 1¾

Figure id, comprising panels A and B. shows images of radiographs and pCT images showing the resuits oi a non-humor· primate fNHP) fibula wedge osteotomy model at δ and 10 weeks Figure 15A shows images of 5-week radiographs obtained in a NHP Shale* WedgeFlgBralSA':::' shows images of the fibulas of 4 ieprbsentative NHPs which ragetved BMP-GER in or** limb and WT BMP-2 in the carrtralateraf Smb at 0,6 mg/ml (260 pG tota! BMP da|iv®rad/iimb) at f Weeks,. Figure 1 SB shows uCT Images* of me same limbs «hi© lie large calluses of the BMPeSBR traced limbs compared With the shfetik

Figure iG. composing panels ART fee-^ngff ;s$lfcess {Figure 16B1, and callus bone volume (Figure 16C) of limbs -msm -itfte BMPFg treated carslralatorai limns.

Figure 1 / composing panels A-G. snows radiographic images of Ire healing Over tend hf Shon-human primate'*. (NHP) fibutes treated with BMP-GBR at 0Λ mg/ml and BMP-2 Ιη.:.β^;':€*^ίϋ:-ί^^'ifcb ai i 5mg/mi usss>g a calcium phosphate bused cement us a earner knowing Ihd wMge defect fnodel Figure 1?A, upper panel, shows rssuks ior NHP number 1 reft arm treated with 0,5 m.g/mi GER as tefbwa; panels I end 2 show LAT {lateral) and AP (anterior-postonc* 5 images. respectively, at the: Initial time poms, panels 3 and 4 show LAT and AP images, respectively, at 2 weeks, panels 6 and e show LAT and AF images, respectively, at 4 weeks, panels: ? and 8 show LAT and AP «mages, respectively, at G weeks, panels 9 and 10 show LAT and AP images, respeci;ve;y; at 7 week?/, panels 11 and 12 show LAT and AP Images, respectively, at 6 weeks' Gcura t ?A Sower panel, shows results ter NHP number 1 right arm beated with 16 mg/mi 8MR-2 as lo-orm panels 1 arse a' show LAT {lateral} and AP (anterior-posterior) lhegesyres|secliveiy, af the iniual torn» point, panels 3 and 4 show uAT enc AP images, respectively. at 2 wamai fmm S and 8 show LAf and AP images, respectively, at 4 weeks; panels / and 8 show LAT and AP images, rasppctiyeiy, af 6 wseks; pahsis f and 10 show LAT: and AP images, respectively, at 7 weeks; panels 11 and 12 show lAT and AP: images, respectively:. at 8 weeks. Figure 1 (B shows the

radiographic the results lor Νί-tP number 2 as described for NHp bl in Figure 17A, and figure 17Ό sets out the resets for NHP number 3 as described tor NHP #1 In Figure 1 7A

Figure ΐβ ts >s diagram of a structural modes showing representations sod compaction of the crystal structures BMR-E and BMF’-δ WF Fhe differences in lito length ol the glycan resolved is highlighted showing that fids gsycao for BMPF mat :s resolved is; much longer she;·, that for BMP6 The; indicates that the BMF’B glycan is more conionnaiionaiiy constrained than that u! BMP6 such that snore of· the glycan can be rendered In this model. Tnc hodsime doorstop residues for both BMPE and BMF8 are shown m ssmsisr non'doorstop configurations Also, the arginine glycan "tether' sis blissing the SMPE giycan Is shown by dotted Sines, representing the interactions of the arginine with the glycan.

Figure 19 ss a closer view o; She histidine doorstop and arginine tether sol the BMFE and S3MP6 comparison shown in Figure 18. This image shows the similar conformation of the HS4 histidine residue of BMRE and the equivalent histidine of BMP6 both in the non-doorstop position The image also shsws the R;6 tethering I via interactions o' the BMPF. glycan such that the glycan is more rigid and therefore mom rs rendered By the modes compared to hie more lloppy” and less constrained giycan of BMPS such that less ol the 8MP6 glycan is; visualized in this model. The· diagram of this modes also shows the similar poulfAonmo of asparagine N66 ol BSvfPB showing N-iinked aftachmant of the giynan and the equivalent and similarly postponed asparagine of BMPS. The diagram also illustrates the potssntsai additional glycan tethenng interaction of BMPS' £110 shown by dotted lines between the ammo acid residue-and the distal end of the giycan. Π» differences :n toe length of the giyeati resolved is highlighted showing that less of the darker 8MPG glycan can ho resolved compared with trie lighter shaded longer glyc&amp;rt tendered for 8MP£ indicating that the BMPE glycan Is mote eonformationaliy cottstwnee end thus more so rendered upon structural analysis. f igure 20 is a gtaph showing the results o' an alkaline phosbatase essay using C2C12 pte-mvohiusis comparing the osteogenic activity of BMP-2, BMPE apd 8k4P-6 with their Fndo-H treated degiycosyfated t'Degly.} counterparts.

Figure 21 Is a diagram Illustrating die structural model of BMPE showing the location ol the glycan tether at RIO and illustrating the stabilising interactions between the arginine (Pits) anti glutamic acid iE110 corresponding to E109 o' BMP21 residues. The diagram shows that Rio and E1"0 both form mull.yie hydiogeo bonds wild the thud iiVn-iannose) and teudh {,s-monnoee} glycan moiehes. The diagram also shows the position of H54 potential 'doorstop' and asparagine 56 (Nob) which provides the N-linketl attachment site ol the glycan.

Figure 22 is .a graph showing the results of an aikaiine phoshatase assay using 02C12 pre-myoblasts comparing the osteogenic activity of 8MP-E, with BMP-E-NR, BMP-GER and BMP-GER-NR in the presence of increasing doses of Noggin - a natural Inhibitor of BMP-2. The data demonstrate that 8MPGER-NR comprising sequences derived from acIrvin was not inhibited by Noggin even a! high concentrations buf that BMP-GER was sensitive to Noggin inhibition, Thus, addition of sequences derived bam eciiyin caused BMP-GER to become Noggin resistant (NR) Those results demonstrate that hoggin s@na8ye, fo^rng'ioggi^iesfe^ot pR} upon replacement of the sequeooasjonyed Item activln.

Figures 23 is a graph showing the bone score as determined by imrmmohistochemisfry rfHC) for rat ectopic implante truatcd with the indicated BMP at the specified dose, t he date show did! tee hone forming activity of BMP-GBR was greate da» re-mm w'hen the C-iermin&amp;i segoeoda of teo molecule was replaced with a sequence derived from ac»v.n {HR' Thus, tee data demonstrate that BMP-GLR-NR was nujnh less ateiva than BfdP-GRR in vtvo.

Figures 24 is a graph showing the bene score as determined by irnmunohisioehemtetry *1MC) for rat ectopic implants treated with the Indicated BMP at the speidled dose. The data show teal the bone forming activity of BMP-t was greatly dqoreashdv indeed/i was completely abrogated, when the G~ terminal seqaenfes of the molecule was tepteosd With a S8qu»snoe derived from activin (NR).

This teven¾prtv^^::t8l·fkrfSs^^^b^e^f¾c¾^teggneiβ· protein, retem-te to herein as btesigiter : BMP." "designer osteogenic protein” and "designer protein." The designer BMPs of the Invention may correspond to tee amine acid sequences of wild typd unmodified BMP, such as, but not limited te, BMPS, BMF4. BMP§, BMP6, BMP?, BMPS, and 8MP&amp;. in parfidUfar embodiments, tee designer BMPs show altered binding to a type i and/or type 1( BMP receptor when compared to Is corresponding wild type BMP. in further embodiments, the designer BMP thby be modified fe have altered haihiite, irnmunogenteity, or any pharmarokinefic/pharmaeodynamte |BK/PD) parameter when compared to its corresponding BMP.

Mlailm

Unless otherwise defined herein, scientific and technical terms used in odhrteCtibn with ihe : present invention shas have -he meanings teat are commonly understood by those of ordinary sklil In the an. further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall Include the singular. Generally, nomenclatures used in connection with, arte techniques of. cell and tissue culture molecular bmiogy. immunology, microbiology, genetics and protein and nucleic acid chemistry and hyfcncnraiion describee herein are those well known and commonly used in the art.

The methods and techniques of my pi event invention are generally performed according to methods well known in the ah and as described in various general and more specific references that era wted and discussed throughout the pi event specification unless otherwise indicated. Sects references include, o.g,, Sam brook and Russeii, iVto'twttee Cloning. A laboratory Approeah, Cold Spring Harbor Press, Coid Spring Harbor. NY {2001), Ausubei ei at. Cmvenf Protocols fo MoteaJar Biology. John Wiley Ss Sons, NY (2002), and Harlow and Laos Anl&amp;odfes. A Labotefery Manna/. Coid Spring Haicor Laboratory Press. Cold Bpdng Harbor, NY (1330), which era incorporated herein by referencs, Fg^ymgle reactions and purification techniques are performed according to Mbnufacturur's speaffeuttens as commonly accomplished in the art nr as described hereto. The nomenclatures :|p.;ipi^nibfioni-::«|fei and toe laboratory procedures and techniques of. anal vUcas chemistry, syntoete: organic chemistry. and medicinal and pharmaceutical chemistry described herein ere those well knoom end commonly used in the art Standard techniques are used fot chemical rn/mhesos, chemical analyses, pharmaceutical preparation, formulation, end delivery, and treatment oi patients:

As used herein, each of Ida following terms has toe meaning associated with it in this section.

The articles V and ‘an" are used herein to refer to one or to more than one t?.e., to at least oho) of the grammatical abject cf the article. By way of example, "an si emend means one element or mere then one element in this application. Ids use cf tor" means "and/or'' unless stater* otherwise.

Conventional notation »s used herein >q portray polypeptide sequences; the left-hand end of a polypeptide sequence ss the amino-terminus: the right-hand end of a polypeptide sequence is the earboxyt-termfnus.·. As used hatetrt. the twenty conventional amino acids end toeir abbreviations loikm conventlonal usage. See !m;nuno!ogy~~A Synthesis {2nd Edition. E. S. Golub and D. R. Gren. Eds.. Sinauer Assoc;;;;lea. Sunderland, Gass (199'!)). amice is incorponutect herem by reference. As used herein, amino acids are represented by the full name thereol by the three setter code corresponding thereto, or by toe one-tetter code corresponding thereto, as indicated as follows' EMiLDte Iteik^gLQf^· Q0Ed:gMEL.E22§

Aspartic Acid Asp ID

Glutamic Add Glu £

Lysine I. vs K:

Atginins Aro R

Histidine His H

Tyros»ne Tyr V

Cysteine Cys C

Asparagine Asa R

Glutamine Gin Q

Serine Sar S

Threonine Thr I

Glycine Gty O

Alanine Ale A

Valine Vai V

Leucine Leu ,L

isokiycine lie I

Methionine Met fvf

Pmdifte Pro P

Phenyfelanlne* Phe t

Tryptophan Trp W A "conservative amino acid substitution" is one in which an amino acid reudue 1:5 subs! luted by another amino acid residue buying a side chain R group wim similar chemical properties (o.g,. charge or hydrc-phobsdty}. In general, a conservative amino acid substitution vail not substantially change the functional properties of a protein, in eases where two or mote ammo acid sequences elite: from each other by conservative substitutions;, the percent sequence identity or degree of simitarlty may be adius.-ted upwards to correct tor the conservative nature of ibo substitution. Moans for making this adjustment ate well-known to those of skill in ibe art. See. e,g . Pearson, Methods Mol Biol 243:307-31 (1991-.

Examples oi groups of amino acids trial have side drams with similar chemical properties include' 1) aliphatic side chains: glycine, alanine, vahn-a. ieeeiem and isoieucine; 2} alipbakc-hydroxyt side chains: serine and threonine. 3} amlde-centamlng side chains, asparagine and glutamine 4i aromatic side chains: phenylalanine, tyrosine, end tryptophan; 5; basic side chains lysine, arginine, and histidine; 6) acidic: side chains aspartic acid ana: glutamic mud; and 71 sailor-containing sate chains, nvsteme and methionine. Pretarred conservative amine acids substitution groups are. vaiins-ieudneesoieuans, phenylaianine-ryroosne, lysine-arginine, alanine-valine. glutamate-aspanate and asparagme-giutomme

Alternatively a conservative replacement is any change having a positive value m the PA be'50 iag-'Sikeiihaod matrix disciosad tn Gonne? er. at.. Sti&amp;nce 256:1443-1440 (1992). herein Incorporated by reference. A “moderately conservative'’ replacement 4s any change having a nonnegative value in the PAM2S0 log-likelihood mefme.

Preferred ammo acid substitutions are those which, ft) reduce susceptibility to ptoteoiysis. -:2) reduce susceptibility to oxidation, (3) alter feinomg affinity for forming protein complexes, and (4) confer or modify ether physicochemical or iunctionai properties of such analogs. Analogs comprising substitutions, deletions, and/or insertions can Include various mutems of a sequence other than the specified peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino odd substitutions) may he made m ms specified .sequence (preferably ih the portion of the polypeptide outside the domain(s) fanning iritermeleculaf commits e g , out si be of the CORs or the type ; ot type ii receptor binding sites). A conservative amino ado substitution should not substantially change the structural characteristics of the parent sequence (e g„ a replacement ammo acid should not tend to break a heN thek occurs in the parent sequence or disrupt other types of secondary structure that characterizes the parent sequence}. Examples of ad-recognized polypeptide secondary and tertiary structures are dev on bod in AVoieeis. Slruotwoe end Motccotof Principles (Creighton. Ed., W. H f-resmao and Company, New York (1934),1. introduction m Protein fbme.Wo? (C Financier· and .:. 7ooze, ecis , Garland Publishing. Now York N.V. (1991)-, and fhornton et a! Netun·. 354.105 (1991:-. which arc each incorporated herein by reference.

Hie terms '‘polynucleotide”, 'nucleotide sequence'. "nucleic acid" ’'nuckiio add moiecUlqG '‘nucleic acid sequence", and "oligonucleotide" refer to a series of nucleotide bases (else catted "nucleotides'') m DMA arid RNA and mean any chain of two or more nucleotides. t fie polynucleotides can be chimeric mudur-as or derivatives or mnblfied versions thereof, sinqks-sttanded or doubie-siranded, The oiigowcieotide can be modified at the ba.ye moiety. sugar moiety, or phosphate backbone., for examplei. to improve siafeHhy of Me roofecoie. ms hybridisation parameters* etc. A nucleotide sequence typically carries genetic information including the information used by cellular machinery ίο make proteins arid enzymes. These terms include double- or single-stranded genomic and cQNA, KNA, any -synthetic and geneficaiiy mania-slated polynucleotide and both sens* and antisense polynucleotides This also includes nucleic acids containing modified bases, To? example, thic-urscll thic-guanjne, and ituor c-uracil. or containing c«rl>ohydfate or lipids. in the context ot a nudeotiee sequence, she term "substantially identical" is used herein to refer to a first nucleic acid sequence- that con isms a sufficient or minimum number of nucleotides, that are identical to aligned nucleotides in a second nucleic acid sequence such that the Hrst and second nucleotide sequences encode a polypeptide having common functions! activity, or encode a common structural polypeptide domain or a common func-sonai polypeptide* activity. For example, nucleotide sequences having at least about 85%. 90%. 9i%, 92%, 93%, 94%. 95%. 95%. 9?%. 93%. or 99% identify to a reference sequence.

By "designer BMP nucleic acids." and grammalica! equivalents herein ss meant nucleic acids that encode designer BMPs,

The terms "protein" and 'polypeptide'' are used interchangeably herein, These terms refer to a sequential chair; of ammo acids finked together via peptide bonds The terms include one or more proteins trial function as a discrete unit, if a single polypeptide is the discrete functioning unit and -does not require permanent or -emporary physical association with ofh*i polypeptides in order to form me discrete lunctioning unit. the terms 'polypeptide’' and "protein" may be used interchangeably, if the discrete functlanat unit is comprised of multiple polypeptides that physically as.-sociaie with one another; the term “protein” as usee herein refers to the multiple polypeptides that am physically coupled and function together as the discrete unit. A protein to b* expressed according to fhe present Invention can he a protein therapeutic. A protein therapeutic is a protest that has a biological effect on a region In trie body on which it acts or on a region oi the body on which it remotely acts via intermediates. Examples of protein therapeutics are discussed m mere detail below "Designer BMP," as the term is used herein, relates to a BMP protein comprising a; least one ammo acid mutation compared to a corresponding vvild type BMP without fhe imitation, whereto fhe designer BMP has detactably altered binding for at least a Type I receptor and/or at least one type II receptor compared with tie binding of the corresponding wild type BMP for the type I and/or type II receptor.

By "corresponding wild type protein" 4 m meant the wild type version oi' the designer BMP prior to the introduction oi any mutations. For example. If the designer BMP is a designer BMP2, iris corresponding wikMyps BMP Is wild-type BMP2. Thus, to one embodiment, design of a designer BMP carp but need not, begin with a wild type BMP sequence wherein mutations (e g., amino acid substitutions. deletions, and/c-i· insertion) are introduced Into the wiki type sequence Therefor*, th* designer B'viP can conespond with a wild type BMP, nob the locations of the n'misbom car; be said, for instance. So correspond with, oe relative to and Or bo respective with the -armno acid sequence of the wiki type corresponding ot reference" BMP sequence

Fite proteins of the present inyuntion include fragments: derivatives. analogs, or variants of tie polypeptides described hereto, and any combination thereof The terms, 'fragment." "variant' 'derivative' and 'analog” when refer ring te proteins of fftc present invention include any proteins which retain at least some of the functional properties; of the protein from which a was derived

By the ten's drags ι-οηΓ its coed heroin refers to a polypeptide and is defined ns any discrete portion of a given polypeptide ihat is unique to or characteristic of that polypeptide The term as used herein -amo refers to any discrete portion of a given polypeptide that retains at least a fraction of the activity of the fuiMengfh polypeptide in certain embodiments the fraction of activity retained Is at least 10% of the activity of the fuis-sength polypeptide, in certain emhertiaieats. the fVactfen of activity lefained ;s at least 20%, 30%. 40%, 50%. €0%. 70%; 80% or 90% of the activity of the falMength polypeptide. In certain embodiments, the fraction of activity retained is sf least 06%, 96%, 9?%,, 9S% or 99% oi' the activity ol the FuSMength polypeptide, in certain embodiments, the fraction of activity retained Is 1009:., or more of the activity of the hdHengid polypeptide Alter noisveiy or additionally, the term as used herein also refers to any portion of a given polypeptide that Includes at feast an established sequence element found in the full-fength polypeptide in some embodiments, the sequence element spans at less; about 4-5; 10. 15, 20, 25, SO, 35, 40. 45, 50 or mure ammo acids of the full length polypeptide, fragments of irrctsms; of the presen: invention include proteolytic fragments, as well as deletion fragments.;

Variants of tfie proteins of 11¾1 preseni invention include fragments as described above, and also polypeptides with .altered ammo acid sequences due to ammo acid substitutions, deletions, or insertions Variants may occur naturally or be ace-causally occurseng. Norvnsiuratiy occurring variants may be produced using air-known mutagenesis techniques Variant proteins may comprise conservative os noo~ conssrvaiiys smirto add substitutions. deletions or additions.

The proteins of the invention Include proteins having cue or filers residues chemically denvafized by reaction of a foncisonai side group .Also included as proteins of the invention are poiypeplKies that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids For example. 4-hydraxypfoSme .may Pc substituted for praline, b-hybroxytysine may he substituted for lysine. Γί-methyihistidins may be substituted for histidine homoserine may be substituted For serine: and ornithine may be substituted tor lysine. 'Recombineoily expressed polypeptide'· and ’recorn binant polypeptide" as used herein refer to a eelypephde expressed From a host cel: thal has been manipulated to express that polypeptide in certain embodiments. Hie host cell is s mammalian coif, m certain embodiments, ties manipulation may comprise one or more genetic modifications; Tor example the host cells may he genetically modified by fhe I shod cotton of one or more heterologous genes encoding the- polypeptide to be expressed. The heterologous, reoombinahtly expressed paiypeptxie can be Identical or similar to polypeptides that are normally expressed In the host ceil 't he heteroiogous rscombinamiy expressed polypeptide can aiso b® foreign to the hoot ceil. s,s. heteroiogous to polypeptides normally expressed in the host cels. In certain embodiments, the heterologous reoombinanttv expressed polypeptide is chimeric. For example portions of s polypeptide may contain amino mud sequences that are identical or similar io polypeptides normally expressed in the host ceil, wnsie other portions coni a in ammo acid sequences that are foreign to the host cell. Additionally or alternatively, a polypeptide may contain ammo acid sequences horn two or mere different polypeptides; that are both normally expressed In the nest cels Furthermore a polypeptide may contain amine acid sequences iron? two or more pehpeptides that ore- both foreign to the bos* ceil, in some embodiments the hast oat; is genetically' modified by the activation or u pragmatism at one or more endogenous genes.

Calculations of homology or sequence identify between sequences (the terms are used interchangeably herein) are performed as follows To deforming tfo.percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are· aligned for optimal comparison purposes (e g , gaps can be introduced to one nr Path a first arid a second amino add or nudsiu nerd sequence for optima I alignment and non-homofogous sequences can be disregarded for comparison purposes) in a typical embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%. at least 40%, at least 50% or 80%. or a? least 70%. 80%.. 80%. or 100¾¾ of the length of the reference sequence The ammo ao;d residues or nucleotides at corresponding amino add positions or nucleotide positions are then compared When a position in the first sequence is occupied by the same amino add residue or nucfootkfo as the corresponding position iri the second sequence, dree te® molecules are Identical at that position (as used herein amino acid or nucleic acid •'identity*' is equivalent to amino acid or nude!·::, acid "hamoSagy1').

To determine the percent identity of two amino add sequences or of two nucleic acids, the sequences are argued for optimal comparison purposes (e.g., gape can fee introduced the sequence of a first amine add er nucleic add sequence for optimal alignment with a second amino acid o? nucleic awe sequencer Ins percent identity between the two sequences is s inaction of the number of Identical positions shared by Urn sequences fi.e . % homology-# of identical positions/tota! ?" of positions X 100). The determination of percent homology between two sequences can be accomplished using a mathematical algorithm. A preferred, norHsmitmg example ei a metfiemescai eigen torn utilised for the comparison of two sequences is tire algorithm of Karim ef si,, Proa N&amp;H .Acad Sci U S A 87 2284-8 11930), modified as in Karlin ei at., Prac N&amp;fi Acad So/ U S A 90.3871%? {1993). Such an aigarithm Iwlncorporafod Into the NBLAST and XBLA8T programs of AltschuI et a).. J '.Ίο/ Baji 215.400-10 (1900), BLAST nucleotide searches can be performed with the NBLAST program. score-·. 190 wordiengih -12, BLAST protein searches can be performed with the XBLASi program, scste-SO, wofdfength-S.: To obtatn capped alignments, for compassion purposes. Gapped BLAST can be utilised os described In Aftscbui et a! , iVuefem Acres Res 23 3389-402 i199?5 Wtien utilizing BLAST and Gapped BLAST programs, the. deiauit parameters of the le&amp;peclive programs* (ex; . XBI..AST ami NBLASTi can be used sequences Is a tugcsfeq of the qumher of id®r>tleai ipositiorm shared by the sequences, taking inld: account the number of gaps) and the :iai#b of each gap, which:; need to be inirndueed for nprimaS alignment of the two sequences.: the defreunination of be accomplished using a mathematical algorithm In one amhedimenh the pmm identify ^tween two ammo acid sequences is determined using the NycdiemamWunach algorithm (bieedieman et at., J Mot Sint 48:443-5¾ f1S?0)) which has been incorporated into the GAP program in the GGG software package uvaiSable on i;i gcg.com), using either a Biossum 82 matnx or a PAM2SG matrix, and a gap weight of 18, 14 12 10, 8. 6 or 4 and a length weight of 1. 2. 3, 4, 5. or 6. in yet another emhudiment, the percent identify between two nucleotide sequences ss determined using the: GAP program in the GCG Sdfhvare package {evaliubie on the internet at qcg..com), using a NWSgapdna.CMP matrix and a gap weight of 40,. 50,60. 20, or 80 and a length weight of 1, 2.3,4. 5, or 8 One typical set of parameters |amf the one that; should be used unless otherwise specified) are a Btossum 62 sconng matrix vwth a gap penaity of 12, a gap extend penalty of 4, and a -rameshift gap penalty of 5-,

The percent identify between two amino acid or nucleotide sequences can be determined using the algorithm of F Myers end W Miliar (Myers e? al.. tMmputAppi &amp;'oac.>4;11~?(1^08)} which has been incorporated into the AUGN program (version 2.0). irsing a PAM120 weight resdue tahie, a gap fehgtt? penalty of 12 and a gup penalty of 4. "instructional material,' as that term is used herein, includes a publication; a recording, a diagram or any other medium of expression which can be used to communicate the ussftdhasp of the compound. combination, and/or composition, of the invention in the kit; for afeoitng, alleviating or treating the various diseases or disorders recited herein. Optipnaify, or affafmafeiy, the insintotihhaf materia} cap describe one or more methods o! alleviating the diseases Or disorders in a csli, a JsSpd, or a mammai, including as disclosed elsewhere herein.

The instructional material of the Kit may, ior example,: bp Mixed ooplpcund and/or composition of the invention or be shipped fe^ether Mh a container v^ieh dpntains tip compound and/or eomposSiors. Alternatively, fhe mstructiortefrpater# rri^.&amp;/sftpped separately front the container with ihs inionllon that the recipient uses the insPucSonal material and the coprgeund: copperaflveiy.

Except when noted, the terms “patient" or "subject·· are used irderdbangeabiy andrefer to mammals such as human parents and nombuman primates, as weil Ps veterinary subsets such as rabbits, rats, and mice, and other animals Preferably, patient refers to a human, 'Effective amount', or therapeutically effective amount," as the terms are used fniiemhangeabiy herein. is an amount the! when administered to a tissue mediates a cetaciabie ihsrapeufic response compared to the response detected ip the afeende of the campAUhd. A therapeutic response, such us. but not limited to. ir^ifhltiorfofafKi/ordocseased fibrosis. Increased bone mass ar bon% deo&amp;lty, and the Hke, ¢80 be readily assessed by a plethora of ^t-i^osgr<i^d including, c.o., suci:- methods as disclosed herein

The skilled arfi&amp;an would understand Inal the affective amount oi the compound or composition administered heroin varies see can he readily determined based on a number of lectors such as I he disease or condition being treated, the stage of the disease, the age and neaith and physical condition of the mamma! being treated, the seventy of the disease, the particular compound being administered., and the like

As used herein, to "treat" means reduces the frequency withTWhfob symptoms of'a disease (e.g,:: decreased hone (tensity. fracture, fibrosis, and the tike) are experienced by a patient The term includes the administration ot the compounds or agents of the present invention to prevent or delay thb onset of the symptoms, complications, or biochemical indicia of a disease, alleviating the symptoms or arresting or inhibiting further development of the disense, condition, or disorder Tieatmeot may be prpphylacfee {to. prevent or delay the onset of the disease, or to prevent the manifestation of clinical· or subdinicai symptoms thereof) or therapeutic suppression or alleviation of symptoms offer the manifestation of the disease.

By the phrase 'soeoifiouiiy binds," as used her#r»f JjSfmtiafctiiles^psMbd, » g., a protein, a nucleic acid, an antibody and the like, which recognizes and bshdp aspecific molecule, but does not substantially recognize or birtd other molecules In a sample For instenbs, dh BMP protein, an antibodyor» peptide inhibitor which recognizes sms binds a cognate receptor (mg., a BMP type I or type If receptor:, an. antibody that binds with its cognate antigen, and the like} in a sample, but does not substantially recognize or Thus, under designatedcondibohs, the speci^i binding moiety (eg., a BMP or a receptor binding fragment fher^sf) binds pi^farentlatiy to a particniar target, molecule and does not bind in a significant amount to eiher components present in a tost sample A variety of assay formats may be used to select an ahfi.f>0dy that speoifroatfy binds a molecuie of interest. For example, sblld'phese EUSA immunoassay, immunoprecipitatiuii, BIAcore, FACS, Octet, ami Western blot analysis are among many assays that may be used to identify a BMP that specifically· reacts with a BMP receptor Typically, a sprrcifie or selective faction will be at mast hvico background signal or none more preferably, ar least five-fold greater than background signal or noise, and more typically, more than 'tO times background, even more specifically, a BMP is said to spepficaliy bind1' a BMP receptee vkhen she equilibrium dissociation constant (Ku) is &amp; 10D pM, more preferably < 10 μΜ, even more preferably S f μΜ, yet mom preferably s. 100 nfv1 and most preferably e ID nM.

The term "toy refers to the egu|ibhum: dissoefaion cemstant of a parties interardion. “Binding affinity" generally refers m the strength of too skitototal: pf hphebyafeht. intoractibhs between a binding site of a molecule (e.g.. a BMP iigand) and its binding: partoer |e,g.( a BMP type l or type It receptor). Unless indicated otherwise, atouspd herein. te.;tejj$p£ic· binding affinity which reflects a Irl interaction between members of a binding pair (e.g., BMf> and its cognate receptor). The affinity of a molecule X for its pprtrKsr V can generaiiy he represented by the dissociation constant (Kd).

Affinity £aa'^i^^wM'by-'Cx»nmon methods known in the art. including those described herein. stowly and tend to dissociate readily, whereas high-affinity BMPs generally bind a receptor fester end tend to remain bound longer A variety of methods of measuring binding affinity are known in the art, any si which can he used for purposes oi the present invention. Specific illustrative embodiments are dasenbed elsewhere herein

Tims term Xw" ns used herem is intended to rater to the association or on rate cbnstant, or specific reaction rate c-f the forward, or complex* form sag, reaction measured in units: iyi" see'.

The tern> ns used herein, is intended to refer to lire dissociation or of; rate constant; Of specsfic reaction rate, for dissociation of an antibody irPfri The antibody/ankgen complex. meashfp in units- sac :

The term *K,H. as used hereto. <s intended to refer to therdissDGteflbh^cidfteishi·of a pariculaf antibody-antigen interaction I- is calculated by the formula k.^' ir.\, — rs,

The terra 'akered binding” ns used herein means the <tesi$j#:0f$> specificity for at ieest a type i receptor and/or a type ii receptor When compared with The binding of: a corresponding wild type BMP to tha same type i and-or type i? receptor. The design# BMP may bind with greater or lesser affinity with the receptor compared to the binding of the wild type BMP to that meeptor For instance, if the wild type BMP hound a certain type I receptor with M certainlanding affinity, the corresponding designer BMP binds that receptor With greater or lesser affinity compared, with the wild typeBMP. it may even he that the designer BMP wiii specifically/:hind f fecaptor that the Aid type BMP did not detestably bind end vioa-awersa vvttere the designer BMP MS hb longer defeotabfy bind a receptor ihai the wild type BMP hinds. Thus, altered binding encompasses any detectable change In btodipg by a designer BMP to a type I or type Si receptor compared with flte.^nidteg"#0tet corresponding wild type BMP. It may be that the designer BMP has a greater or ibsser k;,ft vaide compared with the kQV value for a; oorresponding wild type BMP and/or the designer’ BMP a greater hr lesser k^- value-compared: with the value of toe corresponding wild type BMP such that toe Kd of the designer BMP ts greater or lesser than the tot of a corresponding wild type BMP for the same BMP receptor Thus, any difference in a binding characteristic and/or affinity value between a designer BMP and a corresponding: wild type BMP are encompassed by the term “altered binding* as bsed herein.

The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that ,.$tov# tteA.#§e---3fcaly6i8 td ^Nfeb'' by: detection of alterations in protein concentrations within a btosansprmatrix, tor example using AB Uppsala. Sweden, and Piscataway, N.J.}. For fortf-ter descriptions, see. e.g.. Johnssop, ef ai., Am. £« din 51: 19*26 <1993); dohnsson. et ai„ matectmqvm 11: δ2θ-62Τ <1991); JohhSson, of al„ duMbP; Recognit 8; 125-131 pfPSto}: and Johnnson, ef ai., Ami. Meehan. 198: 368-2.77 (1991),

As species fs-ifepredotninant epecies present (i.e., on a molar basis it is more abundant than any other Individual species in the composition), and preferably a substantially purified fraction is a composition wherein the p|j|fof species (s.g., a designer BMP) comprises at least :'5#j#|^lj^roent (on a molar haisss) of eii macromotecufer species present Generally, a .substantially' pure composition will comprise more than about 80 percent of all mecromosecuiar species present in the composition, more preferably mom -ban about 35%. 9U%. Bibs. and 93%. Most prefers»!v\ the object species u< punffeb to essentia! homogeneity (contaminant spades cannot be detected in fee. composition by eorn'entsonul detec,bon methods) wherein the composition consists essentjasy of a single msoromoiecuiar species

Bono j^orpboPanefio fimtoins {BMPsV

As staled previously elsewhere herein, BMPs are members of fee TO'F-?> protein superfamiiy aii of which ons cbaiaefehsert by sbeocnsfefvad cysteines residues (Lender ette. (2001) Nature 40¾ 860--921 The BfeP/GDF subfamily includes, but is not limited to. SMP2.8MP3 (dsteogsrtin) (sea, s.g. US Patent bio 6,177)406). BMPSfc iGDMO) (see. eg , US Patent No 6 204,047), BMP4 (BteP2b) (see, e.g US Patent No. 6,245.839}, BMPS (see, e.g., US Patent No. 6 5*3,394). 8MP6 (see, e.g., US Patent No. 8.813,744). BMP? (osteogenic proteln-i or QP1) (see. e.g.. US Patent No, 5,141,505), Bfy5F5 tOP2}feee, e,g„ US Patent No. 5.688,6/3), BMP8B (QP3) (see. e.g.. US Patent No. 5,854,011), 8MP8 (GOB};^/· s.g„ US Patani No 6.287,810} BMP10 (see, e.g,, US Patent No. 5,703,043), BMPII (ί?ΦΡ11) feee.e^,, US Patent No, 6,437,141), BMP12 (GDh?) (see, e.g., US Patent No. 6,027,019), BMP13 (G0F8. GDMP2) (se« eg., US Patent No 8,027,919), BMPf£$3J3f&amp;) fse#f e^„ US Patent:Jo, 8j34,229T BMP16 (see, e\g„ US Patent No. 8,331,642)., ^4^2¾ m *&amp;βφη .No.:^04^^1^|K;|^P3. fees, s.g, US Paten! No. 6.026,475). GOF5 (CUfePI; MP52) (see, e g., US Patent No. 6,924,084), and GDF8 {myooUiisn) (see, e.g., US Patent No, 5,627,733). BMPs specifically· hind their cognate receptors, wbfeh inclube Type I receptors;:: ALK-t, ALN-2 (ciso bailed AetRla or AcfRi| SLIPS (also called BMPRte), arte ALK-6 (alee caiied BMPilb): fend Type If receptors. ActRI :a (also sjled ActRif}, ActRlifo, and BMPRfb The BMP-recepfer binding ihfefaclons have been studied esttensivefy, type BMP for each iypb Tandfer type If receptor is generally known, in the art and are shown in Table 1, See, e.g.. Nickel etai,, Cytokine Growth Pastor Rev 20:387-77 (200% Heinecke etei,, SC Biot7:591200S). TABLE!

This application Is based, in pari on tho uifoersianding that each BMP dinner hinds To four BMP receptors twa type I receptors two type H rscepf^ of each BMP for each receptor arc known in the art as shown above in fabie 1, φφν foe rocbpr binding Mpona of various BMPs that madtofe binding of the BMP for each receptor have been mapped and are shown In Table 2. For instance, it is well established that wiki type BMPS end BMfM· bind type ! BMP rcvfopforv Aik-3 and AlK-B vwto high affinity and bind type ii 8MP scepters, with lower affinity. On the other hand, wild type BMPS and BMP / are known to have o*od type ii reeepbrs ActdlA, ActHB, and BMPRIi wifi high affinity but bifid type i receptors, with tower atiiniiy than they do to lype II. it is beleved thai the differing cellular fosp^see from foe approximately forty^three TGFJ5 snpedhmfly foidmhecs signairng through inforaeion with approrimateiy twelve receptors is believed to be due io each ligand whirring a epepitfo repertoire of receptors with which =t hinds vwth differing affinities. The type ;i il feinting d«mat!&amp;#!e Table 7. TABLE 2

Ratfonaf airite iacid stfoshttnion to after receptor hinriino of desigper BMPs In one embodiment, foe invention comprises introducing an amine acid mutation so at least one receptor binding site thereby providing aliened binding is type i and type is BMP receptors, by designer BMPs compared to toe binding of the corresponding wild tyjre BMP to those receptor Thai is it Is wsif known in toe art that wild type 8MP3 shows a relatively high affinity for type \ receptors, white wild type BMPb shows a high affinity for type si receptors it « farther known tn the art foot hsteroOimeto of wife' type BMP2 and BMPb bind to both type \ and type It receptors with relatively high affinity each BMP apparently providing the higher affinity binding site each raoepte Bee Table B, feelow. The 8MP2/b heterodimers are known to be more active that BMP2 or BMP6 alone or as homodimers, in both in vitro and in vivo bone formation assays. Table 3 shows an example of BMP2 and BMP6 binding affinities to type I and II receptors. TABLE 3

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.

Accordingly, the invention relates to provide designer BMPs with improved binding to type I and/or type II receptors. As shown in Figure 1A and Table 2, each BMP comprises three binding sites that contribute to receptor binding. From N- to C-terminus, each BMP comprises a type II receptor binding site A, a type I receptor binding site, and a second type II receptor binding site B. Although an exemplary alignment of wild type BMP2, BMP4, BMP5, BMP6, BMP7, BMP8, and BMP9 is illustrated in Figure 1, the skilled artisan will appreciate that there are well-known alignments providing the relative positioning of various amino acids among the members of the ΤΘΒβ superfamily. Such alignments are provided, among others, in International Publication Nos. WO 2009/086131 (e.g., Figures 15-17, Figure 31 A), WO 2008/051526 (Figures 9-12), WO 2005/118636 (Figure 6), WO 2005/118635, WO 2005/113585 (Figure 3), WO 2001/92298 (Figure 6A-6C), Kirsch et al., EMBO J. 19 :3314-3324 (2000) (Figure 1), US Patent Application Publication No., 2007/0293425 (Figure 6), Groppe et al., Nature 420 :636-642 (2002), Nickel et al., J. Bone Joint Surg. Am. 83:7-14 (2001), and Weber et al., BMC Structural Biol. 7 :6 (2007). Thus, using protein sequence alignment algorithms and tools well-known in the art, including the alignments of the amino acid sequences of the various TGFp superfamily members, as well as the disclosure provided herein, the corresponding amino acid in one BMP/GDF protein relative to the amino acid at any position in another BMP/GDF protein can be determined. In one embodiment, the corresponding amino acid residues in BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8 and BMP-9 are shown (see, e.g., Figure 1A).

In some embodiments of the invention, the designer BMP comprises mutations in a type I binding domain or a type II binding domain, wherein the mutations confer altered binding to a type I or type II BMP receptor. In some embodiments, the designer BMP comprises one or more mutations in both a type I binding domain and a first (binding domain A) or second (binding domain B) type II binding domain. In other embodiments, the designer BMP comprises one or more mutations in both type II binding domains. In other embodiments, the designer BMP comprises one or more mutations in the first type II binding

domain, if; the second fyps; IS binding domain, and m the type S binding domain M some embodiments, the designer BMP comprises one or more mutations in She type i binding domain.

In some emfe&amp;dimsnts. the mutations in'; prove binding to a type 1 imoeptor. in other embodsc'ients, it;e mutations improve binding to a type i| receptor in other emhodi;cents, toe mutations decrease hindleg to a typo I or type li receptors % some emDadimen'iS tine mutations create or destroy a giyoan tether as mete fuby set forth below. In some embodiments. the mutations create or destroy a rbs doorstop as; more fully set forth be I rev.

Because BMPs are so well characterised and understood in the art. 4 would be understood, once-provided with the disclosure provided herein, the location of possible mutations that can be made that do not further affect the activity ot the designer BMPs would be understood. Accordingly, the designer BMPs of the invention encompass variant BMP's which dlffsr from a corresponding wild type or designer BMP in fbai it contains additional insertions, deletions, or substitutions which do not affect me receptor isocline affinity of the variant BMPs, in some non-limiting embodiments, those o! skill In the art. would understand that the cysteines involved m cysteine bnot formation and amirsa acids mvafved irt rrsceptar interacfiorits should not he mutated or should he changed with conservative substitutions;, while other amino acids may be more treeiy substituted, omened or deleted without adversely affecting biological aciiyify of the designer BMP. it should be noted that unless otherwise stated, at! poi5st.loi-.el numbering of designed or modified BMPs is based on the sequences ot the mature native BMPs. Designer BMPs are characterised by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic pr interspecios variation at ihe BMP sequence. Variants of designer BMPs must retain at least 60% of the activity of trie corresponding wild type or designer BMP nativity in one or more cell types, as determined using an appropriate assay described below. Variants that retain ai least .'5%, 80%, 80%, 90% or W% of wild type activity are more preferred, and variants that are more active then wild type ere especially preferred. A designer BMP may contain insertions, deletions, and,'or substitutions at tbs N-- terminus, C-terminua. or internally In a preferred embodiment, designed or modified BMPs have at feast 1 residue •bat differs front the most similar human BMP sequence, wilt; at feast 2, 3, 4* 5. 6, 7. 3, 9, 10 or more different residues being more preferred

Designer BMPs of ths invention maintain at least 60%, at least. 31%, at least 62% at Mast 83%, at least 64%, at least 8¾¾.. at feast 88%. at least 8/'%, at least 88%, at least 83%, at feast 90%. at least 31%, a- feast 92%, at least 93% at Urns- 94%, af least 95%, at least 96%, at Mast 97%, atleast 98%, or at least 99% identity with the corresponding wild-type BMP protein sequence.

Designer BMPs ot the invention may rnambsm a* least 80%, at least 81%. at least 62%. at laasl: 83%, at least 84%. af toast 85%., at least 86%. at least 87%,at leas; 88%, at feast 69%. at least 90%, at least 31 %. af least 92%, at teas;· 93% at least 343¾. at feast 95%. at least 99%. at least 97%, at feast 98%, or at least 99% identity with the conserved cysteine domain of the C-torminA region of the corresponding wild-type BMP protein sequence. ;:j3sslsper BMPs way contain modifications, for Instance mutai>on!| that; alfor addliiorsaf protsfo isropertles such as stability or atimunogenioity or which enable or prfevant po^traoslatiohai modifications such as, PEGyiation or glymsyiafion. Designer BMPs may he subjected to so- or past-translational modifications, including but not limited to synthetic dortvaiizatton of one or rnoro side chains or termini. giycosyfeitjon. PFGyiation, circular permutation. cyd&amp;ation, fusion to proteins or protein domains, and addition of peptide tags or lands.

Duo to the degeneracy of the gen esc coda, an extremely large number of nucleic acids may ho made, ail o! wisen encode the designer BMPs of the present invention. py simply modifying the sequence of one or m^nr o <00 o, ir ,3 oav 'hot rot G' nc* the amir ,» >' seguer-e <0 too desi> 51 or BMP T no designer BMPs of the invention do not comprise these sequences sot forth in VVO2Q0d;0o1S2S of WQ2Q09/0861 i'f 1,

As deseeded above, BMPs are naturally sttpiassbd ,m: pro--pi«fote compri^fig. a; long pro-domain,, one or more cleavage sites, end a mature domain. This pre-protein is then processed bv the cellular machbi^Y to yratd a: - BMPs of the invention are pm>dyced: in a similarmanner.The profoofnein is f^iisved to aid ip the comaet folding and processing 0? BMPs Furthermore, in some Pu! not all Bi#s, the pro-domain may noncovasenily hind the mature dornasn and may act as a cAaperontyas well as an inhibitor (e.g., Thies et a! (2001) Growth Factors, UV25l-2hB) Preferably. the modified BMPs of tt® invehtfen are pr^uced

AtemativeSy, BMPs may he produced tnrofo^· forms, including, but not limited to. where the mature domain is produced directly or refolded from inclusion bodies, or composes fulHengfh intact pro protein, i'he de$g^M#>a. of^VffW^bn wiiibo tsscfoi in thass ano other forms.

In padioular embodiments, the designer BMP of the inydnilon comprises a backbone BMP i.e. fos w8d type BMP. to which the designer BMP oonespohds. ih particular embodiments, this backbone BMP may ho a wild type BMF2. 8MP4, BMPS, BMPS. BMP?, BMP8, or BMP9 backbone. in some embodiments of foe invention, the designer BMP comprises at least one mutation in a type I binding domain and/or a type h binding domain, wherein the mutation confers altered binding to a type I or type if BMP receptor compared with the binding of a corresponding wild type BMP not the deigner BMP comprises at least otfo foufotfon In both a type I binding domain and at least one mutation in a type it binding domain, in other embodiments the designer BMP oqmprteesat ^ It birfoihg domain A and the type il binding domain 8. in other embodiments,, the designer §MF comprises at least one mUfoiion :n type il bindmg domain A. type ii binding domain B. and a type | binding domain.. in certain embodiments, trie mutation may comprise an amino or nucieic acid substitbtfoft, deletion and/or insertion, in a preferred embodiment., the mutation comprises an amino acid substitution.: jo some embodiments, the haftkbon© BMP <s a wild ty$e BMP and the muia^a am one or mors of the mutations iisted in Tables 4 to 6, Ttsajeslgner 'an^ssiirti^r of mutations listed if» these tables

In some embodiments. the backbone BMP is a MM type BfvlP and the tnulatbnsem one dr more of the mutations listed In Tobies 4 to 6 The designer BMP may contain a permutation an$H%«t?<l all of the mutations lish-od In these tables or disclosed elsewhere herein IABLB4

TAB!

F is

TABLE a

in some sntoodimsnh:·, the mutations mfptove binning to a type I receptee to other amfeod'-mants Improve binding to a type I! receptor. In other embodiments the mutations decrease binding to a type i or type ti receptors;.

Tables -f-G above provide a non-timing compilation of example mutations of the present invention where tha position of the mutation is provided relative to the aw-eopcvsAng wild type BMP ammo acid sequence, i ons, in some- embeds;vents. the designer BMP composes trie following preferred Oi'tinbif'fatians of rmjtafkwis..

In certain embodiments. the corresponding vvrld type BMP to the designer BMP is BMP2. Further, toe at least one mutation within the type it receptor binding domain A &amp;a niititeilid^.^ti^ied from the group consisting cl V33. P3S, G3/, H39. v4 t: Y42 AND H44. in other embodiments;, the designer BMP comprises at least one moradon within the type |i receptor binding domain A and further comprises at least one additional mutation within a type Preceptor 'binding domain. The mutation within the type t receptor binding domain is at least one mutation at Pf£\ F49. A52, D53 H54, L.%, Νί&amp;. S57. Ν5», V63, 7Β&amp; Ν88, 88¾ V70, N?1 8¾ K73. I74, m4 WB φ respect ίο the sequence of SEQ ID NON. in yet f-iilher embed imsnis. the designer BMP comprises at -oast one moi.ai.ion witton a type ϋ receptor binding domain A. at least one mutation wiihin the type I receptor binding domain, and further comphs.es at least on» additional flotation within a type OB receptor IhWingt^olwsirt. The mufaflen Within: the type If receptor Diriding domain B is at toast one mutation at E83, 889. A8S, M89, LS2, E94, N9S, E96 K97. V9S. and V99 with respect in the sequence. of BEG ID DO: l i?i sortie eovoexiiments. the designer BMP composes mutations at each of amino acids H44. PAT A62. D63, L55. S3? N68 BBS V70. insertion of P after ΝΠ, S7.2, K7% 174s A77; and V8D with respect to the sequence of SEQ IQ NO. ;. in one embodiment, the designer BMP composes the fditowihc mutations. H44D, P4SS, AS2N, DS3A. LbSM. S5?A: N88H: S89L V70M. inserhen of a P after N71 S72E, K73Y I74V, A77P, and V80A with respect to the sequence of SEQ ID NO:'!. in same embodiments 4¾¾ designer BMP composes mutation?; at each of amine acids V3&amp; P3S. H39. SS5. M89.1.93. E'34, E36. K97. and V99 wli'h respect ίο the sequence of SEQ ID NO:1. in some: embodiments, the designer BMP composes mutations at each of amino acids Υ33ΐ, P3SK: H39A, S85N. M89; I.S2F, E840, E96S. K97ST and V99I with rsspsci to the sequence of SEQ ID NOD. in oilier embodiments, the designer BMP composes the following mutations: V33I. P36K. H39A, H44D. P48S. A52N. t.fMM, S5AM. N6SH V70M. S7?R, K73F, insertion of s Y after K7S, 174V. 77AP. S8SN. M89V. L92F, E94Q, ES6S. K9rN and V99t with respect to the sequence ot SEQ ID NO:'!, in yet other embodiments. the designer BMP comprises; the following mu Prisons: V33I. P36R. H39A, H44D, P4SS, A62N. L54M. SS6M. N68H. V70M. 372L. K73E, insertion of a Y after K73, i?4y. 77AP. SB5N. MS9V. 1.923. E94D, F969 K97N. and '3991 with respect to the sequence of BED ID NOD in certain embodiments. the corresponding w*ld type BMP to the designer BMP is BMP4. In certain embodiments, the at least one mutation within in® type M receptor binding domain A is at V35. P38,7339. 041, P43. Y4a. and H46 of SEQ 43 NO 2. in other embodiments, the designer BMP4 comprises at least one mutation within toe type is receptor bidding domain A and further comprises at least one additional! mutation within a type I receptor binding domain. Tbs mutation within the type f receptor binding domain is at least one mutation at P5Q. AS*, D5S. H56. IS?. N58, S59, NCD. VCD. 767. N70, S71, V72, N73. 874, 878, f7B. and P?7 of SEQ ID NO;?. in ye; further erni>odimenfs. the designer 3MP4 co opposes at least one muishon within a type 11 receptor binding domain A. a; leas! one mutation within the type I receptor binding domain and further comprises at least one additional mutation within a type 08 receptor binding domain The mutation within the type M receptor binding domain Q is at least one mu tailors ai E8S, So A ABB M91, 1.34. ESS, Κ8Ά V98 and V99 of SEQ ID NO:?. in certain embodiments the designer BMP is BMPiF In certain embodiments, the mutation w»fh<n the type il r^^{^:ft^iriQ,dbfifjairt':A i&amp; -si: legist ah£:m$aid!n4&amp; 156, Γ-59. GSO. Ah? FG4, YGri. or DG7 of SFQ iD ND:3:.

In other embodiments, the designer BMP comprises at least; ode mataioh Within the type it receptor binding domain A end farther comprises at feast ope additional mutation yjrf^-:$4^ji{gee|jfcr‘ Wudihg domain* the mutation within the type t receptor tindi^dbiwh is at feast MM?f,:; FT?. N75 A?G. H7? M78. N?9 ASQ. N8? V8G T88. «91, IM, MS3* P94. P9S; 0¾ «97, V98, or P99 of SLQ ID NO 3. in yet further embodsments. the deiogoer BMP edm^i'ses at least one hlhtafjon wlfm a; type; If receptor binding domain A. at least one mutation within the type I receptor binding domain., and further oampdsas at least one additions! mutation within a type !!3 receptor binding domain, The tfiaMidh within the tvpe ii receptor binding domain B is at least one mutation at K107, N109, Al 10, Vi 13. F116, D116. S119. StaO. N12I. V122< or !123 ot SEQ ID ΜΟ:3>

In certasn embodiments the comieponmng «did '.BMP In oartain embodiments, the mutation within the typed! receptor feMJf^.#jiR^''i||%'Mlea^ i£i7# :KS0 G81, Λ63. Mob. Y6$, or DG8 of SEQ ID MO:4 <n other embodiments, the designer SMFA oesbfS'isas at least one mutation vyMtlih the type if : scepter binding domain A and additional mutation y#hin a type i recaptgr binding domain. The mutation within the type t robeptor fethdihg domain te at toast one mutation at #7i,. N7G. A?7, H78. M79 M80, A81. «83, V8? Τ8Θ. «92, 1.¾. M94, N9S. P96, E97, W,W, or PI GO of SEQ !U HO A. in yet funner embodiments: the'.dfisigmsr i06|^''''fphs^ises gt least one mutation within a type it receptor binding domain A, at least; one niutatiorr wittim the type I receptor binding dornaini and farther comprises at least one additional mutation within a; typs IIB receptor Ending domain. The mdfatlon wijfein; the type il receptor binding domain B is at least one mutation at -Kl-iiS*;Ni $%.;A1 tt, V114,: FI 17, Pi IB. N120, S121, N122y V123, or 1124 of SEQ ID NQ:4, in certain embodiments, the corresponding wild type BMP to the designer BMP Is ΒΜΒΤΓ In certain embodiments, the mutation within the type if receptor binding domain A is at least one mutation at m.YBS, or E68 of SEQ ID NG.b in othei' embodiments, the designer BMP? comprises at least one mutation within the type it mpoptor binding domain A and further comprises at feast one additional mgtatioh within a lype I rsoepfor binding domain. The mutation within the type i receptor binding domain is at least one mutation at A?2, F73, N?8. S77. Y?8. M?S. N8Q, A81. N33, V87. T89. «92. F93. !94, N9*>. P9B E97, T# VB9, dr ΒΙΟϋ Of SEOIDHOtS.

In yet fuilher smbs^diments. the designer BMP? cenrprises at least one mutation within a type il receptor binding domain A. at least one mutation within the type ! receptor binding domain, and further comprises at teas! one additions! mutation within a type l|B receptor t>in<!ing dom» The mutation within : lihsi: ΪΙ bindididbmAi >a ai ·*Α OfSS muts8oh^Μ G108; N110, At IP V114, E117, D119: mm. Siai. WS^m oril24«f S£QiDNO;S, in certain embodiments, Ihe corresponding wild type 8MP to the designer BMP is BMPS In certain embodiments, the mutation within the type h reesptor binding domain A is at feast one mutation at 167, Q60 G6t. 863. V8S Y88. or E6S of SEQ ID ND'fi. in other embodiments, the designs BMPS comprises at feast one iAfMbn Within the type it receptor binding domain A and further comprises at feast on* additional mutation within a type i reosplor binding domain; t he mutation within the type t receptor binding dgmA that least one rnutation at BTt , F?3. D76, S77, C78. M79 N80, A82 M%. I 87. S8I, 1¾ »4, M#,: P&amp;6. 037, A3S or F10d ot SCO ID NO-8.

In yet further embodiments. the designer BMP8 ddfnpNads at teais| &amp;nd· fhUJalfejh'· tiritftfcf #%pf h i'eeeptor binding domain A, as least one mutation within the type i receptor binding domA, nod further comprises at feast one additional mutation within a type I SB. rSoe|^rb^it^:^ip^MhAtTTe''md^d^ within the type ii receptor binding domain 8 >e at least one imitation at K1O8. St iO. A111. VTi4. Ydi7; 0118, Si19; S120rMl2f. N122. V123. or 1124 or SEQ ID,$CM

In certain embodiments the mutation within the type Si receptor l^ri^,dorpi»ii. mufetion at 127. K30. E31. ESA Y3S. or E36 of SEQ ID NO·:?,"

In other embodiments, the de&amp;ignei BMPS comprises a; least one mutaliorl within the; type tl teoepipr binding domain A and further composes at least one additional mutation within a type I redeptor binding domA> The mutation wilhin the type t: reoepiar binding domain is at least one myMioo af F42> P43. A4S. P47. D48, V49, ISO, PS1, K5S, V57, tbP, H62, 163, K64, F6S, PS6, TW, KoS, VSS, or G/Ό ot SEQ ID btO:7.

In yet tbhher embodiments, the designer ©MBS' comprises at least one m dfptfeh within A type i t recaptor binding domain A. at teas* one mutation within the type i receptor binding dAA, and forfher comprises at least one additional mutation Within a type HB receptor binding domain. The mutation within the type II receptor binding domain B is ot least one mutation at K78, SSO, P81; VB4, K87; DS9, fv19Q, G91, V92, P93, or Ϊ34 ol SEG ID NO: 7.

Exemplary amino acid sequences of designer BMPs are sbtT^Srtlh til Table 7; b^oWv Table 7 shows the name and seqtienee of the designed molecules.

Tmmii

Althougl i the a Dove II skid designer BMPs comprise cm see scents c? the if iventson, the invention ss not limited in any way hs any specific molecules. instead, rha ioveniian encompasses any design*? BMP comprising altered re seel nr binding where tne designer BMP composes at least one mutation within a type Is receptor binding domain A, even more preferably, the designer BMP ci-ropriaea at least one further mutation within a type i secepio? binding domain most preferably tne designer BMP css;prises yet another at least one further mutation within a type II receptor binding domain B, in other embodiments, ids designer BMP si the pmaeei invention comprises qp amino ac?d sequence at least, about ?G%, 75¾. 8C)'%. 85%, 87%. 90%, 92%. '35%.. 36'%. 97%, 99%, 995b or klsmSoat to ess of the sequence:* deseeded above In another embodiment ttie designer BMP comprises an amino add sequence at toast about 70%. 75%. 80%.. 85%. 87%, 90% 92%. 95%., 35%.. 97'%. 98%, 99% or idenycai tp the sequence of 8EG ID NOs.8-75 in yet another embodiment. ttie designer BMP comprises an amino acid sequence as set forth in any one of SeQ ;D MOs'8-73. in another smbodimsnf, the amino acid sequence of the designer BMP consists o! one of she sequences of SfcQ ID NOs:o-?3,

Further, in one embodiment. the designer BMP comprises an amino acid sequence a- leas? about 705¾. 75%. 80%. 85%. 87%. 90%, 92%.-. 95%, 06%.. 37%. 987¾ 9951.. or leectk.q! to the sequence of SbQ. ID MO 12 In another embodiment. the amino add sequence is the sequence of SFO ID NO' 12. in yet another embodiment, the designer BMP Is BMPE. in an additional embodiment, the designer BMP composes an amino acid seqqenop at least about 70%. 76%, 80%. 85%, 87%, 90%. 92%. 95%, 989% 97%, 98%. 99% or identical to ftp sequence of

SGQ SO NO'14 in another embodiment, the amino odd sequence is the seqiienee hf SESJ IP NO;14, inf yet another embodiment, the designer BMP ;s BMPG

In another embodiment the designer BMP comprises, an amino add sequence at least about 70%, 76%, 60%. 650¾. 8/%. 9038, 98%, 95%, 96%.. 97'%, 98%. 995¾ or identical to the sequence of ShQ ID N0 36 In another embedment. toe amino acid sequence is the secateocs of SEQ iD NO-38. Its yet another embodiment. the designer BMP is DMPGE.

In another embodiment, the designer BMP comprises an amino acid pequuncu at least about 70%, 75%, 80'%. 85%, 87%, 903% 985-.., 955¾. 06%, 87%, 983¾. 99% or identic#to the sequence of SBQ: !D NG3? in another smhoriment. tne amino add sequence is the sequence of S*EQ ID N.D'37. in yet another embodiment, the designer BMP is BMPGER A designer BMP of the invention may' compose a fragment of any one of the Sequences described above in an embodiment, a designer BMP fragment may compass a fragment of at least an uninterrupted 20, 52. 24, 26. 26, 27, 26. 30. 32, 33. 34 36, 38, 37, 38, 40. 41, 43. 44, 45, 4?. 50, S3, 54,: 58 >5 % V' oc nd 70 7' 71 77 SO 83 75 8b 90 <->' -13,98,97 99,100 052, "05, 106, %D -13 tin, 117, 1:9, 120, 12% 122. or 125 amino acid sequence from the sequence of any orte of the sequences of SCO ID NOs 8-73 ii is welt known in the art that BMF’s are -a^m^&amp;Hidbr carboxyl termini of the protein That is, the present invention cornphses; a designer BMP comprising an ammo acid feist 10 amino acid residues, preferably. 9 amino acid residues, even more preferably. 8 amino add residues, yet room preferably, 7 amino acid residues, preferably 6 amino acid residues, even mere preferably, 5 amino add residues preferably 4 amino add residues, more preferably 3 amino acid mpidfies. even more preferably 2 amino acid residues, and^ mdsf pafefebif : 1 aminoadd reside ffem the C and or N temifnus of the designer BMP. in another embodiment the invention comprises a designer BMP protein comprising an amino add sequence of any one of She sequences of SECT ID NO:8-73 and further comprising a ddeioh/lrunaation from if to amino andfef ear boxyi termini of the pfefein; In another embodimehi, the invention comprises a designer BMP protein derived from a BMP protein comprising an amino acid sequence of any of tie sequences of SEQ iO NGs S‘73, wtlareih the protein comprises an amino add dsletion/lruncaiion at the amino and/or carboxyl terminus comprising a deletion of at least 10 amino acid feddttea, preferabiy, 9 amino acid residMes, even more pmferabfy,6afeinp add fesiduefe yet more preferably, 7 amino add residues, preferabiy 6 amino aad residues; even mom preferably. 5 amino acid residues, preierably 4 amino add residues, more preferably 3 amino aoiu residues, even more preferabiy 2 amino acid residues, and most preferably I ammo amd reside from fee C and or N terminus of Ihe designer BMP protein amino add sequence Π;* data disclosed herein demonstrate foal BMPS homedimars pfocSusad In P, coil b^Fsifi a? Έ cds BMP2"), which are not glycosylated, are less active than glycosylated BMP2 produced In roammaiien rails, such as CHO ceiis {referred So herein as "CHO BMP?"}, in addition. data disclosed hermiti further demonstrate that £ coii produced BMP6 homodimerv are cssenilaiiy non^ndlonat compared W:th BMPG homodlmem produced in mammalian ceil culture.

The data disclosed hero»· demonstrate that there are significant variations In the cryMal straefure ot F. eoli BMP? competed with CHO BMP? In foe tvpe I receptor binding report

In one embod-rnsni, the designer BMP comprises m. 0m$: ·Λ^ΐ3ιί^|,;:% glycosytaftou thereby affecting a binding motif that, in torn, mediates altered Madinp to a type i rooeptor. This is based on foe present discovery that In mammalian'{e.g„ points towards the receptor interface white foe HS4 points'tsway' froi# foefe©eptofe: This is in contrast to E cosi-proclucad BMP? where the 053 residue points away from -jtf*e HS4

residue lines up toward the receptor, stacking: against a (tfofine ''r0^e:":;s»'"'illUstraW in P%yrd 3. opparemiy acung os a “doorstop.” In addition, foe dote disclosed heroin demonstmto for the first lime that CHO-produced BMP6, which is fully giycosytated and active, ate comprises a histidine residue pmhting toward the incoming receptor. i%i-:a:-hfstjd«^ tooomtopT

Without wishing to be popod the data dtsoiosdd herein suggest, for the first time, that moving a 'doorstop" residue away horn the receptor interface, coo mediate increased binding between the BMP ligand »d Its fedeptbr. foal the dobrslop: reerdue may be either mutated itself to remove foe doorstop or other residues nwy be mutated to shift the position of the doorstop residue. Purifier, foe data disclosed herein further demonstrate that other residues may be mutated la provide a “giyean tether" which then, in rum, can; orient a giyeari sdeh that the tethering of tl *; ylycan will f cement the doorstop residue.

Therefore, m tamo embodiments, a designer BMP can tm ammo acid mutation that affects the glycsn tether and/or remOve#,^:N#8rfW''d,^^bP structure thereby providing a designer BMP with altered receptor binding.

In summary, m some embodiments, the designer BMPs of the invenlidn may cmftprise at feast one mutation In the type i and/or type II binding domains of BMPs that confer altered type t and/or type If receptor binding fo one embodiment, foe BMP sequence is engineered to alter the receptor affinity of BMPs in order to alter and improve the receptor binding and/or osteogenic activity of the engineered of designer'' BMP in one embodiment, this engineering involves PenMyihg foe residues involved in type I and type ii receptor binding and replacing them to create designer BMP molecules thai show, among outer things, high©* affinity to both type : and typb; it^receptors than the parental BMP horn vsfotoh the designer is derived in other embodiments, the designer BMPs of the Invention comprise mutations that create a new arginine 'giyean tell-e·'' or destroy on o-sfng one to reshape the type I receptor binding domain t nut is. tiie mutation to an arginine id the position two residues.- C-term-mi from the first cysteine, equivalent to R16 of 8MB2, appears to cause ih«s giyean chain to be dethered' onto the BMP surface and consequently alter the conformation of the pre-heilcal loop region compared with the wlid type BMP that tacks the smstatkani In. other embodiments,. the designer BMP of the invasiiion qiay comprise at teas* one nsutatton that alters, creates Of destroys fatjoiishes) residua that Nooks type t receptor trom further engagement with BMP That is, the mutation of HS4 m the designer Bbt>o or a corresponding equsvaiont residue tharMfythat is^oriented^ M eueh a way fed ft impedes or increases interaction of the designer BMP with a type f receptor. in some embodiments, the amino acid mutation affects the conformation of the designer 8MR such that the mutation mediates the creation and or abolishment of an arginine :'giyoan tether* otherwise present m the corresponding wsid type BMP, In some emhodimehtsydfe rnatation mediates an altered: conformation which creates or removes/aboiishes a histidinedoorstop conforiaatieri ifi the designer BMP where such «joorsiop cooteariattoo is to« corresponding wiki

type BMP

Therefore, the skilled artisan, once armed with the teachings, provided herein * would- appreciate that the presence or absence of an arginine "Qiycars letheri· and/or a histidine 'doorstop* in a TGFp euperiamity member may be assessed using any method: known in the art for the structural analysis of proteins, including, but not limited to. tee methods exernpiifled herein. Once the presence of a "doorstop" ίestoue has been identified, then of least one mutation can be introduced info the moteepte to regrient the histidine away from toe receptor binding interface A}tentetoteiy<\« imuto8on that wsii create or enhance a 'glycan tether* such that the Inhibitory aifsct pf the histidihe ’ttcorstep’·, if present h decreased or, more preferably. siiminated.

In one embodiment. where die "GRp snperinrmiy member Is BMP2, the mutation that removes the histidine doorstop is substitution of another ammo acid tor H54. ,tet.'eP.h^''i^ebodk]ntMto« the HS4 is replaced with alanine glycine serine, or threonine

Although the present (nyendoti d:scioses such ''doorstopV«mtoving mutations tor SMP2, the skilled artisan would understand, based on toe knowtedge in the art. hPW to identify corresponding mutations for other TGFg. superfamiiy members end readuy produce mbtooteJacking a "ddofstPpp' i.e,:. removing or reorienting a residue ihsi woukf otherwise interfere with receptor binding by toeing or protecting into: the binding ih^rface. the- effects of toe mutetion on pnjfein conformation can he determined using any ari-recophiaod method for the structurai anaiysis to, Those disclosed herein, Alternatively, mutations that can rernove the doorstep and increase ligand binding to the type i receptor eMibd -using computer modeling methods available in the : art: -Therefore, the present sriveniign ericonipasses the design of T<3Fh appariamiiy members having improved binding With toe: type i: receptor in that they tack a hlstidtoe “doorstop0 residue that Wouid otherwise he present in the receptor interface. 'T^-.pr^^t:fnvenbip;'^^pf:'ph&amp;V!d^i to® sktiied artisan with the understanding of how: to identify mutations for other TGFp family merobsm that would generate or destroy the arginine glycan tether. tether- te^ protsm Sac^Rp the tAsr srp cente^j^ied by me instant invention. Therefore, the present Invention moampmsm- the design of TSFp supertamlfy members having improved binding with ihe type I receptor in that they contain an arginine giycan tether that alters the eontormabOn of the type 1 receptor birthing domain. fn seme embodiments, the removal of the histidine doorstop thereby removing the requirement of a yiycan tether, provides a designer BMP that can ho produced Without giycoeylatson while maintaining hioioglea) nativity. For example, dftsigiisr δ hi Pa may be produced In celts with qlyoosyietion activity that differs from mammalian ceils or is not present, such as bacteria; ceils, yeast ceils, insect ceifs. or slime mold rails In particular emticrumenis. the designer BMPs may ns produced in F coil and maintain biological activity.

Thus, m some embodiments, the invention provides methods for designing and producing BMPs, that can be produced m ceils either lacking giycosytation or composing altered giycosylahon such that an altered glycen Is produced which differs from that produced by a mammalian ceil. Thai is, the present invention encompasses methods for introducing a mutation that removes a doorstop residue that weuid otherwise impair or inhibit receptor binding. The skilled artisan would understand once provided; with the teachings of the invention that a doorstop residue that impinges upon the receptoMigehddp^rfeee'lriiay-. be mutated to entirely remove the residue or other mutations can be introduced such that the residua is oriented away from the Interlace. Such other mutations Include, but ere not limited to, prgypng e giytssn tettier that will alter the conformation of a glyoan and thereby alter the conformation of the ligand Pilch that the doorstop residue is orientated away from the binding interface.

The invention also includes nucleic acids encoding designer the BMPs described hefeih, fTbcfefc acids encoding the designer BMPs described herein can be prepared 3Ρ(^η}ιης:;;:^.^;·Ιίΐί^|'. f$^o*p:|jf -¾ methods known in the art.

In one, nucleic acids encoding designer BMPs are prepared by total gene synthesis or by site-directed mutagenesis of a nucsnc acid encoding wild type or modified BMPs. Methods including tern plate* directed ligation. recursive PGR, cassette mutagenesis, site-directed mutaoenesis ar other techniques that are weil known tn the art may be utilised {see fcr example Stmthe* at at., P>oc Nail .Read Sci. USA 93,16012-1501; {1995}. Prodromes and Fed. Prof, eng. 5: 52/-8.39 (T393r, javarsman arid Puccini. Bfctechqtques IP: 392-398 (Ιδ9% and Chalmers at ai„ Bfofqchmque* '3D: 249-25? 12001)5

Thus, embodiments of the present invention can comprise nude^c acid molecules that encode the designer BMPs of the present invention, tn certain embodiments, the invention piovides a nucleic *kt molecule that encodes for one of -I is amino acid sequences of ShQ ID NQs,8 to 66. fh other embodiments, the nucleic acid mofeoule encodes, a designer BMP protein that comprises :an amine acid sequence at least 70%. 75%, 50%. 35%. 87%, 30%. 92%, 93%. #1%, 35%, «%, 37%, 98%, 939k Idenficai to the amino acid sequence of SFQ ID NO:12. in some emispdimpris, jhs ntlcisfc acid molecule encodes o designs BMP protein mat compm es the amsno s®pysncs: of SE^ JQ·; NO: 12. In another embodiment, Ih* nucleic acid molecule encodes thsamtnq acid seqMepceM BMPE as: set forth *n Table 8. in other embodiments, me nuctefc acid poiecuie obicodes a designer protein Pat comprises an amino acid sequence at least 70¾. 75%. 00% 85%, 87%, 50%. 92% 53%, 94%, 55%.. 96%. 57%. 98%, 99% identical to the amino acid sequence of 5EQ ID NO:14. in sense embodiments, the nuefeio acid rooleeuie encode?, a designer BMP protein that comprise?, the amino ,a^d: ^qiience.: φ$ξ$£· 10 NO 14. in anottser eintxjdirncnt. die nucleic acsd molecule encodes the amino acid sequence of BMPG as sat forth in Tabie 8. lb other embodiments. the nucleic acid moleedie encodes a designer BMP protein that composes : an amino add sequence at least 70%, 7S%. SO1:'.. 85%, 87%, 90%. 92%. 93%: 943¾. 95%, 96%. 97%. 58%, 99% ^ri^i%:i^e.:8mirK> acid sequence of SEQ ID NO:38. in some embodiments, the nucleic add moleeuie encodes a dedgner BMP protein: that comprises the amino acid: sequenca of SEQ ID : N0;3S. in anothbr embodiment Pse nucleic acid molecule geodes the aminoacid^odquence of SMPGE: as set forth in Table 8. in other embodiments; the nucleic acid molecule encodes a designer BMP protein that composes an amino acid sequence at least 70%. 75%, 80%; 85%, 87%, 55%, 92%, 53%, 94%, 95%> 98%; 87%, 93%. 95% identical to the amino ado sequence of SEQ ID NG:37. In some embodiments, the nucleic acid molecule encodes a designer BMP protein that. ceropfises -&amp;M-SEQ' 10 NO:37. In another embodiment the nucleic add rooiecuJe encodes tie amino add; sequence qf BMPGER as set forth in table 8,

Exemplary nucleotide sequences encoding designer BVfPa are se| forp) in Tables, hahM, Table i: S allows the name of the protein encoded snu the nucleotide sequent s ehsOdihg that protein. In generat fhs mature pro-eh coding sequence bagiOsat nucleotide 84? of the sequences iisied below, ΡΙΐ:

ί______________________________________________________________________________________________________________________________________________________________________________________________________________

;l___________________________________________________________________________________________________________________________________________________________________________________________________-

In other embodiments, the nucleic acid molecule encoding e designer BMP eompUses a rudofc acid sequence at least 40%. 50%. 60%. 655¾ 705¾. 755% 60%:s 6S<%; 87%. 30%, S?%. 03%.. 04%. 95%, 965¾. 975¾. 98 .¾. 995« identical to one of the nucleic ac.u sequ»oe^ set forth in SEQ ID NOe 74-139 os <j fragment thereof. In other embodiments, the- nucleic acid molecule encoding a designer BMP comprises a nucleic acid sequence at least 40%. 50% 60%, 655% 70% 75 %, 30% 65%. 67%. 90%, 92%, 935¾. 945», 95%. 9636, 97%, 98%, 99¾. identical to one of the nucts-.c add sequences set forth in Table S or a fragment thereof. tri another embodiment. the oucietc acid moiecuie the nucleic add sequence of any sequence set forth In SEQ ID NOs:?4~139. In yet another embodiment. #ihq nucleic add sequence of any one of the hudde »ld sequehces cf SSQ |D ; NQs;74-l 3a in another embodiment, the nticleie add molecule encoding a designer BMP comprises a nucleic: aad sequence at least 405% 60%, 605¾. 65%. 70%. 757¾. 60%, 655¾. 87%, 907¾. 927¾. 936¾. 947¾. 95%, 9(sv0, 977¾. 98%. 99% Identical in the nucleic add sequence of SPO ID NO:78. or a fragment thereof. In pnother embodiment, ihe nucleic acid molecule encoding a designer BMP comprises the nucleic ecid sequent·» of SE.Q !0 NO 78 In yet another embodiment the nuctsic add moteeui® consists of the nucleic add sequence of SEQ ID NO:'78 encoding SMPE. in another «rcboCsmsm, lbs nucleic acid molecule encoding a designer BMP comprises a nucleic add sequence at least 40%. 50%, 80%, 65%, 70%. 75% 80%. 85%, 57%, 80%, 92%. 80%. 945¾. 95% 98%, 97%, 38%. 39% Identical to the nude;;:. and sequence cl SEQ ID NO‘80, or a fragment thereof in another embodiment the nucleic acid molecule encoding a designer BMP comprises ine nucleic acid sequence of SEQ ID NO'80 :n vet anolher embodiment, the nucleic acrid molsc-iie consists of the nucleic acid sequence o; SEQ ID ΝΟΜΟ encoding BMPG.

in another embodiment, the nucleic acid molecule encoding a designer BMP comprises a nucleic acid sequence si least 40%, 505«, 80%, 5585, 70%, 75%, 60’%. 855:·., 87%, 30%, 92%. 9855 94%. 35%. 96%, 97%, 9885, 93% identical to the nucleic acid sequence of SEQ ID NO902, or a fragment thereof In another embodiment. the nucleic acQ moliscii!» encoding a designer BMP comprises the nucleic add sequence of SEQ ID NO: 102. in yet another embodiment, the nucleic add molecule consists of the euekec add sequence of SEQ SO NO 102 encoding BMPGE in another embodiment, the nucleic acid molecule encoding a designer BMP comprises a nucleic-acid sequence: at least 40%, 50%, 6089, 8559, 70%, 75%, 809% 85%, 679% 96%, 92%, 93%* 348% 95%, 96%, 97'%, 98'%, 99% Identical to the nucleic acid sequence of SEQ ID NO:103 or a fragment, thereof, in another embodiment, the nucleic acid molecule encoding· a designer BMP comprises ibe nucleic seed sequence of SEQ ID NO: 103 In yef another embodiment,, the nucleic· acid molecule consists of Inn nucleic acid sequence of SEQ 10 NO: :03 encoding BMf-’GER, BMPs aro naturally expressed as pio--pfoie;r;s comprising a long prodamaift, doe or room cleavage s;i?es, arid a {nature domain This pro-protein is then processed by Me cellular machinery io yield a, typically, diroene mature BMP molecule, in some embodiments the designer BMPs are produced In a similar manner. The prodoroairs Is believed to play a rose in the folding and processing of BMPs. Furthermore. In some BMPs, the prodomain may noecsmalordly Mod to the roaMm protein and -act as a solubility enhancer, chaperone, or Inhibitor in some embodiments. BMPs may he produced as mature domains produced dlrectty horn or refolded from inclusion bodies. In other embodiments, the BMPs are produced via chemical synthesis or any other known method lor protein production, 1st one embodiment. the designer BMP is; producing using chemical syttfhesis mifihods such as;, but not limited io, synthetic methods weil-knoysfO in the ah, in some embodiments, nucleic acids encoding -designer BMPs are prepared by foial gene synthesis or py she directed mutagenesis of a nucleic acid encoding a wild type, designer, e? variant BMP. Methods Include template directed ligation. POR. cassette mutagenesis;, site-directed mutagenesis, restriction enzyme digestion and ligation. or other techniques that ary vvell known -n the art (see. eg. Prodrome·;.; ef al . Protein Eng 5 627-9 (19:42): Joys reman sf at, Bloteohniques; 12:392-8 i 1982) Chafe's $1 al.. BkHeehniguos :)0 249-.¾ (2001); and SafchioeK and Bossoih in: Molecular Cloning A Laboratory Approach. Cold Spring Harbor Press. Cold Spring Harbor. MV (2001)),

In same endxodimer.hi, an expression vector that comprises a gens encoding a designer BMP 1¾ prepared. Numerous types o? appropriate oppression vectors and suitable regulatory sequences lor a van My of hoof cells are known in -hs art The expression vectors may contain transcriptional and translational regulatory sequences including by not fruited to promoter sequence*, nbosemai binding sites,, transcription»? tarminatnr .signal?., polyadenyiation signals, eric enhancer or activator sequences, in some embodiments, the regulatory sequences include a promoter end transcriptional start and stop sequences In addition, she expression vector may comprise additional elements such as two replication systems to allow it to be maintained in two organisms. I tie expression sectors may be extrachromasornai vectors or vectors that integrate into a host ear's genome In some embodiments, the expression vector contains at least one sequence homologous to a bos! ceil s genome to promote integration into the genome. Constructs for integrating vectors are wait known in the art. In seme embodiments, the expression vector comprises a selectable marker gene to allow the selection of a stably transformed host cell Selection marker genes are well known in the art and will vary vvith the host cell used

The expression vector may include a secretory leader sequence or signal peptide sequence that provides for secretion of the designer BMP from the host ceil Suitable secretory leader sequence?; and sigmii peptides are known in the1: art.

Nucleic acids encoding designer BMPs may be introduced Into host calls either alone or in combination with an expression vector so that the designer BMP is. expressed from the nucleic acid The method of introduction is largely dictated by Use host cell type Exemplary methods of transfsetbiv'franaformafion include C-aPCo precipitation, liposome fusion, electroporation, veal infection, dextrsn-mediaied tranxieciian. polybrsno-meci so Ieci transfection. protoplast fusion. direct micfoinieetion ond other methods known m Me art. Nucleic acids, encoding designer 8MP?< may s,sable integrate into the I lost cell genome or may exist bens:unity or stably in the cytoplasm

Appropriate host celts for expressing designer BMPs induce any cels suitable for expressing wild type nr native BMPs, including, but riot limbed to yeast, bacteria, acchaebsetena, fungi, insect, and animat ceils. In some embodiments the host cell Is Saeoharomyces cerevislae or Eeoheria coil. In some embodiments, the host cel; m a mammalian call such as 283 -eg , 29AT and 293-h’BNAp BHK, OHO (e.g,, CHOK1 and DG44), COS. Jurkat, NIH3T3, or 02012 ceils, Other suitable cells may be found in the ATCC catalog Designer BMPs; may be produced in more complex organisms, including but net limited to plants and animals. In one embodiment, the cells may be additionally genetically engineered, I.®., to contain exogenous nucleic acids other than the expression vector comprising the designer BMP nucleic acid.

in some embodiments, designer BMPs are produced by culturing a host ceil transformed with an expression vector containing a nucleic, acid encoding a designer BMP under the appropriate conditions to Induce or causa expression of the designer BMP. The conditions appropriate for design®! BMP *xpresaiot) are the same coadtfcotw· known to be appropriate for sspraasing native or wild type BMP% These conditions wilt vary with the choice- of expression vector and host celt, and may be easily OS-cor-alned by one skilled in the art through tontine experimentation. la some em bouts not tis, the designer BMPs may be purified or Isolated alter expression. Standard purification methods inoiude electrophoretic, molecular, immunological, and ohromaloqraphic techniques. Including -.on exchange, hydrophobic, affinity, and reverse-phase HPLC chioraatography. and chromatofoossing Csrsxral guidance in suitable purification techniques may be found in Scopes, In; Protein Purification. Springer-Veriag, MV. 3i:: Ed {1934) The degree of puniroatSssh hiibteSiS&amp;fy. Will -vMy depending on the desired use, and in some instances no purification veil be necessary.

Purification from hactonisl ceils may resell tn the expression oi BMPs in inclusion bodies and a subsequent step of refolding in a CHAPS/High sail system Purification from mammalian ceils may Involve n two-step purification vsa Ceilufine-Sultafs and Reversed Phase eimsmaktoranhy columns. in some embodiments, ifte designer BMPs may be modified covalently or non-covafehtiy Covalent modifications may be introduced to a protein by reacting faceted amino acid residues rJ the protein with an organic denvaiizing agent capable of reacting with sstected side chains or terminal residues. Opiunai sites for mndirlcailon car: be chosen using a vaeety of criteria, including but not limited to visual inspection, structural analysis, sequence analysis, and molecular simulation.

In some embodiments, designer BMPs may be lafcatad v-ath at least one .element, isotope, or chemical compound. The label may be an isotopic label such us a radioactive or heavy rsofepe. In some embodiments. the label may be an Immune label such as an antibody or antigen In some embodiments, the label may be a colored or fluorescent label, ouch as fluorescein. It? soma embodiments, the label may be biotin a tag (e g., FLAG, Myc. Hie)

The designer BMPs may be derivslRed with bifneclona! agents to crosslink a designer BMP to a support maids or outface for use in purifying antibodies or proteins that bind to the proteins or to detect binding In screening assays. Commonly used croesitnkiny agents include hut ere no? limited to 1.,1-hss{diaaoaceiyI)-2- phenyisthans, qluiaraldehyde. N-hydroxysuccintmide esters, ior example, esters with 4- astdosaiscyiio rued hemoblfunciionai irntdoesiers. including disacc-oiosny! rioters such as 3.3-dilh;obi.s(succlnlmldyiproplonate): blfundic-nal maielmldss such as fes-N- mateimide-l.8-octane. Other modifications include deamidation of giutaminy! end asparaglnyl residues is the corresfxmdtng gbitomyi and aspartyi residues, respectively, hydroxyfatton of praline and lysine, phosphorylation oi hydroxys groups of scry! or ihryenyi residues, methylation of the ammo groups of lyame, arginine, and histidine side chains ff.E Creighton. Proteins: Structure and Molecular Properties. W. H. Freeman &amp; Co., San Prancfseo pp. 79-8S {1983)). ecseylelic-n of tfie M-terminal amino, and amidaticn el any C- tormina: carboxyl group. Such cieri vats cal ion may improve the solubility, absorption. transput t across tnc blood brain barrier serum nob-life. and the like. Medifsctsbone of designer BMPs, may alternatively eliminate or attenuate any possible undesirable stde effect of the protein. Musettes. capable ot mediating such effects ate dss&amp;osed. fat ©Xitrcpie. in Remington's Pharmaceutical Sciences ibfh ed . Mack Publishing Co,. Easton;. RA (i 989).

Another type·, of covalent modifl/ation c-f designer BMPs comprises kicking the protein to one of a variety of nonproleinaeenus polymers, e.g.: polyethylene glycol ΓΡΕΟ>:): polypropylene glycol or poi/oxyaikyienes. In the manner set forth In U.S Patent Mas. 4,640,835; 4.496 689. 4,301.144: 4,630.41/: 4 /91.192 or <1,l?9;33f. A variety of coupling chemistries η;ay be used to achieve PEG attachment, as Is vvetl known in ihe art.

In another embodiment, the designer BMP from prises linking -he protein vie a CovM-hody linker to a CovX-body antibody such as, but not limited to, the CovX-bodies described In US Paten? No 5,/33.757. strip US Peter:! Publication Ho. US 2009/0098130. Such CovX-bodies may exhibit crip roved characteristics. Including, but not limited to. improved stability and extended serum half-life.

The receptor binding activity' αI designer BMPs may he assessed using any methods used far assessing the activity ot wild type BMPs.

The affinity of designer BMPs ter one or more BMP «scepters can be deiarmmel by receptor binding assays For example, affinities for Ai.K-2. At.K-3 ALK~€, ActRil, AcIRIIb. or BMPRI? can be determined. Suitable binding assays include, but arc not limited to ELISA, fluorescence anisotropy and Intensity, scsntl!iabon proximity assays I SPA). Blaeore (Pearce et aL EMcne/rasey 38.81 -89 (1999)). DFLFi.A assays, and AiphaScrserG'·® (oomroerciaity available from PeridnFimsr, Basse F?.. Illy C. and Chelsky □ {2002)), in some emnodimerifs. Siaeoie or surra·::a piasmo-n resonance essays are used. See. lot example, McDonnell. Cum Opto Chem, Bird. 5,572- 57/ -2001). Biacore experiments have been used previously to characterise binding of TGF-S i&amp;otorms to thee receptors -De Grascenao el al.. J. Bior Chem., 276. 29932-29643 (2001). De Crescendo of a., Mot. Biol. 328, 11 /3-1183) (20-33).

In other embodiments, a Male-based Direcl Binding Assay is used to determine the affinity of one or more modified BMPs for one or more BMP receptors This method is a modified sandwich BUSA in which BMP is captured using an anti-SMB monoclonal antibody and then detected using a BMP receptor-Ec fusion protein

In other embodiments. AlphaServer?'* assays (Boss® R. et aL Principles of AlphaSereen™, PerksnE-tmef Uieraiure Application Mote Ret 44069, |-:Up:Glfese:erices.perluneimer,com/ Notes/S^OBP-0802.pdf (2092)) can be use<1 to characterise receptor and inhibitor binding. Fluorescence assays may also dr: used to characterise iecepio? and inhibitor binding. For example, either SMP2 et a BMP2 receptor or inhibitor -may be labeled with a fluorescent dye dor examples et suitable dyes, sod the Molecular Probes catalog) Additionally, scintillation proximity assays (SPA) can be used to determine receptor binding affinity, for example. BMP receptor-Be fusions may be bound to protein A coated SPA heads or flash-plate and heated with S35aaoelsd BMP fhe binding event results in production of light. {n a particular «w»fcwd»m<shi. the KO of a speeiftc BMP type IS ree^tor pan be determined by using receptor extracellular domain fueibfss toye buman |g:G~Fc. The receptor can he bound lo an octet sensor us«ng anti-hums» vigC-Pc sensors and ihe MP can· bind the receptor extra-cellular domain in solution to determine Kors and Kofi rstoto The Octet systems utilise proprietary Sid. aye? interferometry (El)} to enable real-time. :aosi~i>'«e analysis of htomoMpuiar interactions and to pi evict o information on affinity. kinetics and concsniiation. As protein* bind tttoPptet sphsor the light passing through the sensor has a; wavelength shift that can be measured with a spectrophotometer. The rate ei the stuff is measured as the analyte binds tod semor and wfieh it ieses bthding:.

Methods of Assaying Ostsopanie Activity of de sinner BMP

The osteogenic activity cl designer BMPs may be assessed using any methods need for assessing the achv&amp;y of wild type BMPs BMPs promote the growth nod deferentialto- s d a hdraher of types os cells. Difrerehtlatlpr! may he monitored bslnc. for exempte tuminesf'ence ienooe^c lor asfcalh-e ρηο.νη.Άίν* or -',uo: eoetoc re.scoots such as Atone Blue or PNPP -.Asahlna ot =u : 1930} t..-ρ. Ceu Res., 22'Jmada et at, 11396) i'iktohem &amp;Pf..hy>. Ids. Coovy^o . 22? 31 ?« 322' sor-toko -x e: t s^SSi hie toe 62:2 559-236¾. Cheng et X 3 A' \tc , v ' xx v h »p v o." x a !'x ate s »sN ex> >k to\h >N f\« , ceil-:. in ederoutos embodiments ropcher gone or actuate do 3AK-STAT s;gnai transdu*·lien pathway. .¾ BMP responsive ceil isos contaminc a BTAT~rei.posa.dve repikter such os Gl-P or loesferuse stay bo used rKusanagt. at at. ^fisj Biol. Cali,, 11 ;e5SSd5},Fd· p’8'1^* fit*' Konev 0-¾ a otoeO:» n e >>«-evs"vp *v Λ<<3 'V * * - !g and i tosebborg tShQxs 3. Brel Chem.. 3 A3 23209- 23205

Osteogenic activity m w fee reassured In cell Basso assays such ee aticaliha ntossgtostocs.BRF--fueitorooe. or Atoano red nto:-xotoavou, all of when are described ih Isaacs at a!.. Mol &amp;id<3ctimh 24.1409-14 ??f 301 Os.

Odirogeok. aon'toty may dsn be thiyastidd: In vlmy vlpyati^iopto bone assays <>r maeimalsn bone grewth models. In some embodlmants, osteogenic arptoy is measured db^ rteto-homan pgmale V - > >χχΐ. a s X : u ! *1 xs: ; ! >1» toOvXu 0 s< -4) <- ' f 34 >

Methods tor evaluating bone mass end geality are known {abtoie •.a^.ri^ylhclu^ev hut are got Shitod |g A-rpy dlfl>::itoion· ρχρ· DptyCT; pOCT, chssMcal aralyds, dimity frootlonatlop. ntst '-p'vron' eo^ nstomotto' mvirv any nmvcnom.'e atxelv'' a a? descnbeO foraxasaple >η I ane ot el. j effi. ;y*r. 15:2.105-21 til :?0Q'i}, One assay for determining cortcai txute density ss th* MIorrfCT v ν' >t P ' v ^ v ,,ϊ I to I t ΐ t , POt vX OO '! ti i! <·, ,

Scoop'· ra3lTaO (Bcance Meek: at »\:3j on a lemur.

As? known: Of dffVelPOOff A A?) or ΐή v*ya method for assessing bon* griaA/iAsA®^sA A iissii Ife^sssssss A sn^feogAa activity oi the designer BMPs of the Inypptksg, isM.^

Oessgnyr BMPs ol the present invention may be formulated for udmsnmtrabcn to a mammal preferably a human m need thereof as pad of a pharmaceutical composition The composition can be ad;mnisiered by any suitable means, e.g., parenlerally. orally or locally. Where the designed BMPs ;s to be adminisigred ;:,<: ally, at, oy injection, ?o a desired Issue site, or sysfemiCzsIiy. scab as by miravenous., subcutaneous. intramuscular iniraerbiial. ophthalmic. intraventricular Intracranial intracapsular. in-raspinai, iniraolslemal Intrsperiianesl. buccal, rectal, vaginal, mtrnnssai or aerosol administration the composition preferably comprises an aqueous soiulon The solution preferably is physiologically acceptable such that administraison thereof ία a mammal does not adversely affect the msmmals normal electrolyte and fluid valuing balance The aqueous solution thus; can comprise, eg , normal physiologic saline {0.9¾ NaCi. G.I5M). pH ?-7A

Useful solutions for oral or parenteral system x. administration can be prepared by any of the methods well known in the pharmaceutical arts, oeseHoed, for example. In "Remington's Pharmaceutical Sciences9 iGennaro. A,, ad , black Pub , 1S90, the disclosure of which is incorporated hareiri by reference}. Formulations can include. lor example. potyaiKyiene glycols such as polyethylene glycol, oils of vegetate origin hydrogenated naphthalenes, and the like. Formulations for direct administration. in particular, can include glycerol and other compositions of high viscosity

Riocompatible, preferably bioresorbable polymers. inducting, for example, hyaluronic aeid. collagen, trice lei um phosphate, polybutyrate, poiyiachde, polygiycollde and lastlde'glyeolide copolymers, may be useful excipients to control the release of the designer BMPs In vivo Other potentially useful par enteral delivery systems for the present designer BMPs can ;noiude elhyfene-v;nyi acelale copolymer pari id-as. osmotic, pumps, ire plan labie iniuston systems. arid liposomes. Formulations for inhalation administration can contain as excipients. lor exempli;, inclose or car·: be aqueous solutions containing, for example^ poiyoxyeihyiene-3-lauryl ether, glycochclat* or deoxychclate. or oily solutions for administration in the form of nasal drops or- as a gel to be applied mtranasaliy.

Aliernalively. the designer BMPs of the invention including designer BMP.? and BMPS ideniifed as described herein may be administered orally. For exam pis. liquid formulations of designer BMP’s car; be prepared according to standard practices such as those described In "Remington’s Pharmaceutical Sciences' {supra* Such liquid farms latkrns can then be added to a nevaosqa or another food supplemeol lor aclrnsnislration. Oral so ministration can also be achieved using aerosols of Sbese liquid iormolstions Alternatively. solid formulations prepared using arf-recognlred sewdsifers cart be fabricated into -ablets, capsules or Icutenges suitable for oral administration.

OpUonsily. the designer BMPs can be formulated in compositions comprising means for enhancing uptake o' the protein by a desired tissue. For ex are pie. tetracycline and diphosphonaies (btephosphc-riates) are known to bind to boms miner»!, parsicularjy at zones of bene remodeling, when they are provided syslemsoally ;n a mammal. Accordingly, such components can be used to enhance eeiivory of the present designer BMPs to bone tissue Alternatively. an antibody or portion thereof that binds speed tonify to an accessible substance specifically associated with the desired target tissue, such as a cel! surface antigen, also can be used If deseed, such specific targeting molecules can be covalently bound to the present designer BMP. e.g. . by chemical cross!!?iking or by ust:g standard genefto engineering techniques to create, for example, an ace! labile bond such at; an Asp-Pro linkage. Useful targeting molecules car? be designed, to? example, according to the teachings of US Patent Ho, 5:,001,51.3.

It ;s contemplated also that some of the designer BMPs may exhibit the highest levels of activity In vivo when combined with carrier matrices i s., insoluble polymer matrices See lor example. US Patent Mo.5,266.553 the disclosure of which :¾ incorporated by reference hares! Currently preferred earner matrices are xenogenic, allogenic or autogenic In nature. It is contemplated, however, that synthetic materials comprising pofylactic acid, poiygiycoiic add, polybutyrlo acid, derivatives arid copolymers thereof may also oe used to generate suitable carrier matrices. Preferred synthetic and naturally derived matrix materials, fhesr preparation, methods for formulating them with the designer BMPs yf the invention. and methods ot administration .are. wet! known rn the art and so are not discussed ;n detailed heroin Son tor example. US Parent Mo 5 366.653 in certain embodiments, the designer BMPs car: be admintsferddto the mammal in heed thereof either atone or m combination with another substance known to have a beneficial effect on tissue morphogenesis txampios of such substances therein, cofactors) include -substances that promote tissue repair and repanemllon andor inhibit inflammation or fibrosis, examples of useful ooiaciors for stimulating hone tissue growth in osteoporotic individuals for example include but are not limited io. vitamin 03, calcitonin, prostaglandins, parathyroid hormone, dexamethaaons. estrogen ano IGF-i or {Grit. Useful abactors for nerve tissue repair and regeneration can include nerve growth factors. Other useful cofactors Include: sympiom-ailaviafeg cofaofors, including antiseptics, antibiotics, antiviral and anbfunga! agents, analgesics and anesthetics.

Designer BMPs are preferably formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable, nonioxie excipients and carriers. As noted above such compositions can be prepared for systemic, e.g., parenteral, administration, particularly in the form of liquid solutions or suspensions; for ora; administration, particularly in the ferns of tablets or capsules: or :ntran;iisai;y. particularly m trio form of powders, nasal drops or aerosols. Where adhesion to a tissue surface- is desired, the composition can compose a fibrinogen-thrombin dispersant or other bioadhasiva such as is disclosed, for example, sn PC'" ϋΒ91·Ό93?5, the disclosure ci which U? incorporated herein by reference. The composition than ran be painted sprayer; or otherwise applied to the desired tissue surface

When administered the pharmaceutical composiflc-n of «vs invention. ts typlcaly delivered is a pyrogen-free, physiologically acceptable faro;. Further, the composition may desirably be encapsulated o? Injected :n a viscose form for delivery to the sTa of bone cartilage or tissue damage t boat administration may be au it able for wound herding and tissue repair. Preferably for t-nne and’or cartilage formation, the composition includes a matrix capable of delivering F3MP protein to the site of hone and/or carriage damage. providing a structure for the developing bone and cartilage and optimally capable of being resorbed info the body Such mafnces may be formed of materials presently in use for other implanted medical applications.

The choice of matrix maf-snai :s based on biocornpafshiilfy. blodegradsfeility. mechanical properties, cosmetic appearance and interface properties The particular application of the designer BMP compositions will define the appropriate form station Potential matrices for the compositions may be biodegradable and chemically defined saierum insiaie, iricaieiumphosphate, hydroxyapatite, paiyiectjc add and pot van ft yd rides Other potential materials are biodegradable and .biologically well defined, such its bone nr derma; cotiagen Further matrices am comprised of pure proteins or extnacollular matrix. components. Other potential matrices are oonhkxiegradeble and chemically defined, such as sintered hydroxyapatite, blngiass sisminmsa. or other ceramics Matrices may be comprised of combinations of any of the above-mentioned Types of mater ial , such as polyiactic acid and hydroxyapatite or collagen and tncslciumphosphafe "he bioceramics may be altered ;n composition, such as m eatciuro-oiomleate· phosphate and processing to otter pore size, particle sure, particle shape, and Lvodegradabllity.

The dosage regimen wit! be determined bv the attending phys.ie.lan considering various factors which modify ihe action of the designer BMP protein. T hess factors induce, without limitation, trie amount of bona weight desired in ha formed, the site of bone damage. the condition of the damaged bone, the solo of a wound, type of damaged tissue, the patient's age, sex. and diet the severity of any infection time of administration and other clinical factors.. The dosage may vary with toe type of matrix used ip trie reconstitution, The addition of other- known growth fae-ers. such as IGF I (Insulin like growth factor is. to the final composition, may also effect the dosage. Progress cart be monitored by periodic assessment of bone growth and-'or repav One method of assessing dona growth or repair is by x-ray Imaging and/or CT scanning, among many arf-recognixed methods.

The compositions can bo formulated for parent arm or oral administratis;'! ip humans or orher mammals In therapeutically effective amounts, e g„ amounts which provide appropriate concentrations of the designer BMPs to target tissue for a Fme sufficient to induce the desired effect preferably, tie present compositions alleviate or mitigate the mammal's need for a rnorpliogon-assoclated biological response. such os maintenance of tissue-specific function or restoration of tissue-specific phenotype to senescent tissues (e.g., osteogenic bene tissue) or the inhibition m reversal of a flbrotic response in a tissue,

As wiif be appreciated by ihosb skilled In the art. the concentration of: ft© cbmpouhda described in· a fherapeutk· composition will vary depending upon a number of factors, including the dosage of the drag Ιο be administered. the cbeeBfeel cbafaefeegbcs; |e,g . hydrophobteiiy} of the compounds employed, and the route: .of adhkhi station: The preferred dosage of drug to Be adrnjnisfer«d also Is likely to depend on such variables as the type and extent of a disease, tissue loss or detent, the overall health stales of the parbeuis? patient, the relative biological efficacy of the compound selected. the formulation of the compound, fegmsenee and types of excipients in the formulation;,and the mote of adminlstrafion, in ggnelal terms, the compounds of this invention may M: provided in an aqueous physiological buffer solution containing about 0.1 to lfi% w/v compound for parenteral administration. Typical doses ranges ere from about 10 ng/kg to about 1 Vitfth a-prjef#fed dpso range being from about 0,1: mg/kg tp too mg?kg of body weighl

Therapeutic tides

Designer BMPs may be used for any indication that wild tpe SMBs are useful for ar tor any; method In which a TGF£ superfamiiy member can be used. Designer BMPs are capable of inducing the developmental cascade of bone and cartilage morphogenesis and to induce" ^''i^cHisIlfSitwad signaling pathways. Designer BMPs Induce greater bone augmentation and ,jNi;W'illTtlted to, production of greater bona mass, bone slrffhess and hone

Accordingiy, designer BMPs may be used to induce bone fomvabbn in a lssue:: Also, designer BkiPs may be used to induce pt operation of bone and cartilage in a variety of locations irrfha; body, Ptc example, designer BMPs may be used to repair joints such as kneo, elbow, ankle, arid finger. For example, designer BMPs may be useful for regenerating cartilage in patients suffering from arthritis of other cartilage degenerating diseases. Further, designer BMPs are indicated for treating teare in cartilage: due to injury. In addition designer BMPs are useful tor induoing fegp: growth jfl; designer BMPs are indicated lor use m treating patients difepofebhS ; osteoporosis. or patients In need of spinal fusion or for repair of fho; spine, vertebrae or the Ilka, in another embodiment, the Invention mdudos a method of hone augmentation and/or ropair. Thus, the Invention encompasses administering a therapeutically affactive ssnbuht of a designer BfdF to a site where it mediates, detectable bone augmentation or repair.

In arvother embodiment, the Invention includes a method of inducing or increasing Smad .expression. The method comprises contacting a celt: comprising Smad mediated expression pathway With a designer BMP of the invention.

Designer BMPs ore capable of indue»:ig ihe developmental cascade of bvpne mrxphogenests and rissue morphogenesis for a variety of tissues in mammals different from bona or bone cartilage. This mofphogersis acnviiy includes the aoHsty to «sduce pfMiferatJon ami dilfemntiatioh of progenitor calls, and the ability to support and maintain the dsfterenMed phahOi^kai fofebgH; the· of events that results in the formation of hone, oaiiiiage, non-mineralized skeieiaj and other adult tissues.

For axampte. designer BMPs mey be used tor treatment to proven! loss oi and/or increase ixme mass in metabolic ftone diseases, General methods lor treatment. to prevent loss at and/or increase bene mass in metabolic Pone diseases using osteogenic proteins are disclosed in U.S. Patent No. 5,-574.844. live disclosures of which are hereby incorporated by reference. Designer BMPs may si so be administered to replace or repair bone or cartilage at Injury sues each ae don* breaks, bone fractures, and cartilage tears. Designer BMPs of ihe present invention may be used for periodontal tissue regeneration. General methods tor periodontal tissue regeneration using osier.a;rave proteins are disclosed »n U.S Patent No 5, /'.'33 8/8, the disclosures of which are hereby Incorporated by reference.

Designer BMPs may bs used for liver regeneration. General methods for liver regeneration using osteogenic proteins are disclosed in U.S. Potent No. 5,849,886. the disclosures of which are hereby Incorporated by reverence. Designer BMPs may be used for treatment of chronic renal failure, Genera! methods for treatment of chronic renal failure using osteogenic- proteins ate disclosed in U.S. Psienl Mo. 5.381.404. the disclosures of which are hereby incorporated by reference. Designer BMPs may be used for enhancing functional recovery following central nervous system ischemia or trauma. Genera! methods tor enhancing functional recovery following central nervous system ischemia or trauma using osteogenic proteins era disvSoeaO in U .¾ Patent No. 6.407,060, the disclosures of which ate hereby incorporated by reference,

Designer BMPs may he used for inducing dendritic growth. Gemma! methods tor inducing dendritic growth using osteogenic proteins are disclosed m U.S. Patent No 6,949,505. the disclosures of which are hereby incorporated by reference.

Designer BMPs may be used for' inducing neuraf cell adhesion. General methods for inducihg neural cell adhesion using osteogenic proteins ere disclosed m U S. Psienl No 6,5100,603, foe disclosures of which are hereby incorporated by reference.

Designer BMPs may be users for treatment end prevention of Parkinson's disease. Gane/al methods fer treatment and prevention of Parkinson's disease using osteogenic proteins are d-selpsed in U,8. Patani No, 8.506.785. the disclosures of which are hereby incorporated by reference.

If η; writer. skua of on ordinary adeem to modify the general methods using the modified BMPs of foe present invention for various, therapeutic uses described above. Exemplary embodiments of therapeutic applications of the modified BMPs of the present invention ore further described be tew

Designer BMPs may be used to repair diseased or damaged mammalian tissue The tissue to be repaired ;s preferably assessed, and excess necrotic or interfering gear tissue removed as needed, by surgical, chemical, abiaiing or other methods known in the medical arts. The designer BMPs then may be proyid&amp;d foreefy to -be tissue locus as part of a sfenfe. bmcompotible composition, either by surgical implantation or injection Alternatively, a stente, bincompatible composition containing modified BfvfP-stimulated progenitor ceils may be provided fo the tissue locus The existing tissue at the locus, whether diseased or damaged, provides tee appropriate matrix fo allow ihe proliferation' arid tissue-specific differentiation of progenitor cells in addition a damaged or diseased tissue locus, parffouiany one that has beer- briber assaulted by .surgical means, provides a mowfcogenicaiiy permissive erndfdnntpnh For some bssues, η is envisioned that systemic provision of the modified BMPs vviii be sufficient.

Designer BMPs may be used in prevent nr substantially inhibit seat tissue formation following an injury, if a designer BMP ss provided to a needy injured tissue locus, it unit: induce tissue rnorphogei:ssis ai the locus, preventing the aggregation of migrating fibroblasts Into oon-cjjffersniiatftd connective tissue. The designer BMP preferably is provided as a stenic pharmaceutical preparation injected jfitd the tissue locus within five hours of the mjury. i or example, the designer BMPs may Be used ter protein-induced morphogenesis pf subsUtniiaiiy injured liver tissue following a partial hepatecromy Variations on this genera; protocol may be used for other tissues Trie general method involves excising so essentially nonregenerating poridd of a tissue and providing the modified BMP. preferably as a soluble pharmaceutical preparation to the excised tissue hocus, closing the wound arid examining the sitb of a future dale Lire bone, liver has a potential to regenerate upon injury during post-fetal life,:

As another example, designer BMPs can also be used to induce denfinogsnesis, Tb date, the unpredictable response of denial pulp tissue to injury is a basic clinical problem in dentistry. Using standard denta! surgical procedures, small oreux («·» y - 2 nim i of dental euiptt can be sufgfcaliy exposed by removing the enamel and dentin immediately above the gu lp (by drilling) of ssmpietsefv perferming a i.vsrUai amputation of the coronal pusp tissue. «pdue«*g hemostasis, application of the pyfp treatment, and sealing and filling the cavdy by stance: d procedures.

The designer BMPs; of the invention may be used to heat fibrosis. The fibrosis may be located in various parts of the body and can be of a pedicular kind, for example, the fibrdsia ttfay be located; in tie kidney, for example, fibrosis as observed in giomerulortenephhfss. dlabetid nephropathy. allograft rejection, and HIV nephropathy, in the iiyerpibif e*?hpste..-Shd Veftc-edciusive disease; in the fang, for example, idiopathic fibrosis {andr-^t^mmuha· the side, for example,. systemic sclerosis, fieioids, scars, and -eosHiophrfia^p^]ib.-:syhdh»rtei in the centrai nervous system. f# ©Xampie, intraocular fibrosis; in the cardiovascular $$0ms. for -example, vaaediar rasten-asis; in the nose, for example, nasal polyposis; in bone isr bone mairow. in an endocrine organ; and in the gastrointestinal system. in one embodiment a designer BMP having the binding characteristics of BMP?, or useful modification thereof (extended half life. increase binding affinity for a same or different receptor compared With wild type BMP? resistance to inhibition by a BMP? antagonist, such as; but not limited to. Noggin, and She like} may be useful to treat, ameHdfate or reverse fifesis. That ss, as reviewed recently in Weiskirohen et al„ 20CB. Frontiers in Bsoscl 14 49&amp;2-5Q12, TGFp< mediates a cascade leading to increased fibrosis, iricidding, but net limited transifidn. The fibrosis-iftdtjolng effects of TGFp; may be inhibited or reversed by BMP?. See aiso Loureiro et. a!., 2010, bjephfoL Dial, Transplant. 25:1099-1100. fteSHer* ^^feihi^fspSe.: feo&amp;8$dh£.fraty also be frosted or ameiiorated by administration of BMP4 (see Pegorier el ai , 2010, Reap Res. 11:85), Therefore, the invention encompasses a design» BMP either basso on a BMP? framework and/or iBeorporating tha type I and type ;i mutation? disclosed elsewhere herein, to slier receptor binding and provide a potential useful therapeutic for hes-ment of fibrosis. In a patient in need thereof A fibrofic disorder may be Induced by a number of causes Including: chemotherapy, for e ram pic pulmonary fibrosis resulting from olsotnycln. chlorambucil. cyciophaphamids. methotrexate, rnostine, or procarbazine treatment; radiation exposure whether accidents! or purposelu! as In radiation therapy, fst example. Interstitial lung disease (11.01 resulting from radianon, sn.-10:.-00¾ental or industrial factors nr pollutants such as chemicals, fumes, metals, vapors gases, etc , for example, ILD resulting irom asbestos nr one? ties!; a drug or a oomblnaiian of drugs; for example, anti biotins, le g. penicillins., suilonamldss. etc.), cardiovascular drugs (t?.g . hydralaeirte, beta biockefs, etc.i. CHS drugs (pheoyluin. ohlogttoruanina, sic.) anti-inflammatory drugs. (e.g.; gold salts, phenylbutazone. etc.}, etc, can cause II.D; an immune reaction disorder, tor example., chtonic graft-versus-hust nteenae with derma! fibrosis.: disease stales such as aspiration pneumonia which is a known cause of ILD, and parasite induced fibroses; and wounds, for example, blunt trauma, surgical incisions, battlefield wounds, etc , as in .penetrating injuries of the GNS, in a parhcelat embodiment, designer BMPs with improved binding to type I receptor ALKI2, such as BMPE. may be used to treat diseases related ίο AI.K2.

Mli

Th« invention includes various lots which comprise a therapeutically effective amount of a assignor BMP ot the mvenbuc along with an applicator and instructional materials which desenhe use of the designer BMP to perform the methods of the invention. Although exemplary kits are described below, tl is coniente o? other useful tuts wsll be apparent to the Molted artisan m light of Ihc present dfsutosure Each of tireso kits is induced within the invention. the invention includes a kfi tor treatment to prevent loss of and/or increase bone mass in a metabolic bone disease in a patient in need thereof. The lot Includes a designer BMP ot the invention. The kit further comprises an applicator, including bui not limited to, a syringe, a bene cement mcano devree, and the like, for administration of the components of the kil b a patient. Further, the kit comprises an :<-rteuoi;on;te material ceding forth the pertinent information for the use of the kit to treat or prevent bene mass and/or increase bone mass in the patient

More preferably, the kit comprises at Mast one designer BMP selected from an arte body having an amino acid sequence selected from the ammo acid sequence of SEO ID NOs:8--/3, even more preferably, the designer BMP composes the amino acte sequence of SEQ ID NO:"2. SEO sQ NO. 14. 5EG ID NO.36 and SEQ ID NO.3/. Preferably, the designer BMP is BMPte, BMP&amp;, BMPGE and BMPGER.

The kit cun compose any number of additional theiapeutte agents for 1reatment to preijt^t bone loss sud/or increase bone mass. Such agents are set forth previously and ihOipde tberapeiitic compounds, cytokines, vitamins other members at th« TC,f}t superfo* til·, among many others ; he invention also relates to an article ot meuutactuoi (e.g., dosage font· adapted for i.v. or oral adminiS-oaticri) comprising a designer BMP in the amount effective to prevent bone ioss and/or increase Done mass (e.g„ more than 10 mgrky, at least 15 mg'kg, or 15 rng/kg}. in certain ombd*$fpeni», the article pf manuteum comprises a container or contamers comprising a designer BMP and a iabei and/or idstruefiorw tor use to treat or prevent bone ioss and/or increase· Done mass

The invention also includes a kii to treat or proven; fibrosis m a tissue or organ in a patient in need thereof. The k;i includes a designer BMP of the invention. The kit furthercomprises eh applicator,; Inciuding. but not limited to. a syringe or device tor delivering the protein, a mixing-device, and the iike, for administration oi the components of the kit to a patient. Fuitliar, the kit ootbpases an irsstructfCnsi material setting forth the pertinent information for the uss of the kst to treat or prevent fibrosis In the patient:

More preferably, the kit cons prises at least one designer BMP selected from a protein having an amino nod sequence· selected from the amine «old iD\N0sl0*7£yi§V»« «ifofs ^ef^stttyp the designer BMP comprises the amino add sequence of SEQ: !D NO:12, 8EQ iD NO: 14. SEQ ID NO:3t? arid SEQ ID NO'3? Preferably, the designer BMP is 8MPE. BMP®, BUPOB or SMPOEP.

The kit can comprise any' number of additiOhai therapeutic agents for treetradnt te preveht bone loss and/or increase bone mass or treat or prevent ffordsip. Such agapte are set fdith prevfousiy end include therapeutic comoomws. cytokines, vitamins, other members of foe f GPjS. superfaroify, among many others.

The Invention also relates fo an article ist foanutactufe (e.g, dosage form adapted/fm·Χν. or oral· «dminlsTrafion) composing a designer BMP in the/stfebmini effective1 to preViBf/ijiprie irnmm.

Done mass or ta treat or prevent fibrosis ;e §., more than 1 rngfog, at least f0 pl§?kg, at !es@f IB mg/kg, or 15 mg/kg) in certain embodiments, the article of manufacture comprises p ssonfoihdr or containers comprising a designer BMP and a label anew instructions tor use fo T&amp;iaS. sihdtor increase bone mass or to treat or prevent fibrosis,

The invention is further described in detail bv reference to foe following expenmentai examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus;, foe invention should in no wav be construed as being limited to foe following examples, but rather., should be construed to encompass any and at! variations which dscofOe/eyidbnt as a Jesuit of foe teaching provided herein.

Production anriFuriftoMtouM Production usino hlccvnahun CcH Culture

Recombinant host CHO costs producing and secreting vvlid type and designer BMPs were gsnemted using standard n&amp;comuiuant DNa procedures. Ctondiuonso medium m®··· generated from adherent ceil cultures. Sne%, CHO cells wore seeded »n medium confining 10% dFBS and allowed to grow to near confluence· far 3-4 days. After mis gnow*n phase, growth medium was dfaoenited and mo eeiis were rinsed PBSHeMF^iSsU&amp;sagup^y Si^tofted to a seritm^reo shedium supplemented y«im 200 ug/mf dexfran suifate, 2mM sodium butyrate.. and lOroM HEFES. Ceils were then cultured for 7 days at a temperature of 31*C.<. C«dfftoned med^i^-.fiar^sted ay,$iarl^;lsy' «sing Merlitemg 15.2; uM filtration. Conditioned medium was stored at *2.0 C until purification. in orderW\purify the novel designer BMP moiecutes from CHO celt conditioned media the BMPs were captured by two stops of conventions! chromatography and the results are shown In Figure ft. comprising paneis A-D, Ontydhe; details of the purification of :BMPE are shown Bereih since aii of the other novel designer BMPs were Pdritled in an eashntially simitar manner CHO conditioned medium (CHO CM) (pH adjusted to 8.0 with 1.0 M Iris, pH 8.0) was loaded onto a Caliufine Sulfate column {6omi. 28 yc 12 3cm) that was equilibrated with 20mM MES pH 8.0, The column was washed with 10 column volumes (CV) of 20 rnM The, pH 8.0,10 Cv' tsOmM MES pH 5 8 and 10 Cv' of Buffer A (6.0 tyt Urea SSmM MES. pH b ύ) The BMPs were Muted veto s linear 0-1.0 M NaCs gradient over S CV' {Suffer B~ 8.0 M Urea. 50 mM MES. 1.0 M Nad, pH 5.6). Upon application of a sodrum £ h; odd a gradient, a oread peak oetween cnnnuoFviries of 30 and «5 mSfem characteristic of BMF2 was observer {Figure 5A1 Fractions were analysed by Coomassse stained SDS-PAOE gels and BMP containing tractions were noojed BMPs in frncFom,- were idsntifed as reducible dimers on SDS--PAQE Non-Reduced gets {soft panel of Figure 5B). ihe BMP pools from the Celiulsne Sulfate chromatography step were further purifier! by prepamFve Reverse Phase HPLC on a 10 >· ?5Qmm Vydac 15 pm C8 Column (BoUunt A~ 0.1 % T f- A. Solvent B~ 90% acetonitrile, 0.1 % TFA), With BMP eluting with approximately 32% aewfoniftifa A tracing of the--Reversed Phase chromatography step is shown in Figure 5C the protein was concentrated and soetonimie was removed using a spoedvac and the concentrate was formulated into MFR-169 buffer via dialysis The purified SDS-PAGE, A280 and LAL Assay (endotoxins). A photograph of an HfSthHgdyqM SEfS-PA0£: go! (toft side of Figure 50) and a Reduced 8DS-PAGE get (right side of Figure 6P| showing thd same gel fractions (F13 through F18) to shown, A total of 16 BMP designer proteins were purified to essentially the same ieveis of portly and erpression/poriftoation yields ranging from 0.3-1,4 rog/L CM and the results are Λ^.6ξ) photogmphsa (Eiguto 8).;: itSrtefiy, wild type BMP2 (INT) and d^igngr BMPs 8MP8E, BMPE, EfGSR. BMPD. BMP S8bR. BMP SNE, BMPB. ond BMP-ΕΝ are shown n photographs of a non-reduced gel (Figure FA) and a reduced SDS-PAGE (Figure 68;, and designer BMPs ai {variant of BMFA). aii (variant of BMPA) c (BMPC). hi (variant of BMP!}, he. 1. f, and g am shown in photographs of a nonwedueed SDS-PA6E (figure 60} and reduced SDS-PAGE (Figure 60),

EXAMPLES g§3§ms&amp;ii£Sk&amp;LMigMiiMJ^^

Approximate^ 8080 C2G12 eeite/w&amp;ll in a: SO-wait piaffe were heated wifi'! the indicated BMP ant tire dose indicated, twenty-four hours posllreatmetif. the plates wore processed fo measure alkaline phosphatase which is an ari-reoognbred assay for osteogenic activity. The culture medium was removed, and the plates were washed twice with calotumAmiipnesiurit-iVee PBS 50 μΙ oi 4-Methyhinrbelhfsryi phosphate (4-MUP Liquid Alkaline Phosphatase Substrate Sigma cat. A M3168} was added to each well, arid the plates were incubated in the dark at 37¾ for 15 mim.!fwse. Fluorescence was measured on a Victor juminometer {settings: excitation at SS5nM: emission at 480nfyi: CW lamp energy ai 1130;, 1 second per well Arte: the madmg was compteta. 50 pi of >> protem assay lysis Puffer (/00 rnM Trls-HO. pH 9,8/0 4% Triton X-100; was added to each wait and the protein concentration was determined rising tire 8CA Protein assay (Piatca) following the manufacturer's rnicrepiata procedure. The alkaline phosphatase measurements 'were then normalized to the total protein concentration (i.e . fluorometdo unis per mferogram of protem; As shown by 11¾¾ graph In Figure 7. C2C t a mesaa pre-myoblast celts treated w;th multiple designer BMP ;aoieades showed significantly increased Alkaline Phosphatase activity. as a marker of osteoblast differentiation, compared to treatment with wed type BMP? theavy Hite whh small circles), Designer BMPs exhibiting increased AP activity compared with WT 8MPA included designers BMPA, RMPF, BMPG, and BMPF. Surprisingly, designer BMPtv demonstrated equivalent activity ίο that of the Wild type BMP2.’8 heterodimer (heavy line- with squares}, which rs known to bind both the type I receptors of BMP? and type If receptors of BMPS with hsgh affinity. Designer BMPE is the result of introduction oi the tow affinity type 1 binding region ol BMP8 into BMP?. The extremaly h?gfi activity of the designer 8MPE moieeuis was extremely surprising since it was predicted that BMPE would have low affvyfy binding to tx>th type ( and type If reuepfors. Iritersotingiy. Ihe otner designer BMP molecules, designer BMPA. designer 8MPF, and designer BSvfPG. have regions of wild type ΒΜΡδ that bind ihe type ϋ {high affinity; rsxrepfors of BMPO which nave Msec introduced info BMP? (see Figure IB) and these designer BMPs showed Increased activity compared to BMP?, but not as high as that of wild type BMP2/8 heterodimer {Figure ?).

Ci C12 coils stably expressing the E Pi P-re open se-el cm erst lueiferase reporter (element is from-the lot promoter) were stated at 8000 oeiiii'w&amp;ll of a 96 we is .and treated with the indicated BMP and do» 4¾ hoars poet treatment, me e<$$ were tysed arid tuciferase activity was reed using iite Pmmega Qual-GlQ: assay kit.

The data disclosed herein demonstrated that not only was the activity ot BM2JB eguivatent to that of BMP-kvS in art alkaline pi tosphat&amp;tse assay, it was also eeurvaient sn a BRE-iuciferase assay in C.2C I2 cells as shown in HQ os's 8 Further BMPc derimnstrated approximately 50-30 fold greater activity so the BRE-luciierase assay compared with veld type BMP-2 {Figure Si. Thus the results observed in the BRE-ludferass (8RE-iui:·} assay correlated stm-nglv With those obtained sn the Alkaline Phosphatase {Aik-phos} activity assay ;n this seme seii type {compare F igure 7 and Figure 8). Results Irons both trse Ai-wphos arid BRB-iuo assays .are also shown in Taisia It) for wild type BMP2 and tha indicated designer BMPs,

Without wishing to be bound by any particular theory, these data suggest that trie addition ot At.k-2 ass a high affinity receptor for BtyiPE could be the reason for its increased osteogenic activity. This is because an ALK ? mutation has Been found to cause fibfodyspntets ssaiftcaas progressiva (FOP), a disease where young children develop inappropriate ectopic bone formation Thus, mutation of AS..K-2 binding Is associated with increased osteogenesis and may be correlated to tha Increased -osteogenic activity of Bid PE. Thus, BMPE Is a new class ot BMP molecule with high affinity for the type I receptors ALK-2, 3. arid 6 iid>df'2?..e.t:iLPfdf?.s C2G12 ceils were prated in 6-wels tissue cuifuro plater at a density ot 4 y 10" ueilwAm"' and !.non hated overnight at 37' 0 Inside a o% 00-¾¾ humidified sir incubator After the recovery penod. the culture mediate was replaced wnh freshly prepared osteogenic differentiation medium. Growth Medium containing cOag/mt HasoorBiC add phosphate (F - Ascorbic Acid Phosphate Magnesium Salt a-Hydrate. WAKO Pure Chemical Industries Bal No. ¢)13-12061). p-glyeeroi phosphate ip-Giyceroi phosphate Dlsc-diam sab, fOrnM i'tentebyOrate: Fiuka BloChemiea Cal. No 50020), and 100 r;M Menadione sodium bisulfite fv'itemm KB Sigma Cat, No. 012518), Πιο indicated BMP was added to the appropriate woiis at the deseed concentration Tbs plates were incubated at 37°C for approximately 15 days with medium repkittemeoi every 2 to 3 days. The celts were stained with Me Aiwenn Red stain following the standard published protocols.

As shown in Table 9. beiovy, designer 6MPB >ndu;;wsl mineralisation ot C3H10T-1/2 mouse mesenchymal stem cells to a far greater extent than corresponding wild type SMP2 as Indicated by alit-tann rod stslnlno. That is, as more ini A if ecu seed beiovy, at doses where wild type BMP-2 was unable to Induce mineralization of the C3HtQT-f/2 cells (5. 25. 50. and iDO ng/ml) 8MPE homodtmer induced strong mln-si'ithsatifjn similar to ihaf of the B MR-2 At beferodimer yji as shown m Table 9. Thus, fire alizarin rod staiumg assay f ecu its further constate the results obtained in tbs Alk-phos and BRE-luc assays as disdosed previously herein:. ΊΓ Α £* i

ΐ: Λ

To determine whethe; the stronger osteogenic activity cbcm'ed in vitro by the designer oorrcspoitdttd in similar increased activity in vwo. rat ectopic bone formation assays wenr parfpfmeib Briefly', an ACS (absorbable collagen sponge· impregnated with the indicated total amount of designer BMP m 160 microidsrs of buffer was «mptemed >nto lag hamstring at 6 weak old mate bong Evans rats. More specflicaiSy'. three 8 mm biopsy punched ACS discs were sutured together with non-resorbable silk satis:es The sponges were wetted voth 160 mierohten of the BMP solution containing the amount of BMP indicated in the chart in Figure S foe.. 0.1 pg or 0.5 ug>. The wetted sponges were equilibrated at ;mm temperature for 20 minutes This sponges ware then surgically pieced mto the hamstrings of each rat bilaterally. Each BMP (wiki type and designer molecules) was placed into bath iimbs of 4 rate. Two week5¾ post implantation the animals were sacrificed and the hamstnnps were dissected, placed in 10% formalin and scanned by pCT uweanco Inc.;· to determine the amount ef ectopic bene present, the amount of hydroxyapatite in milligrams (mg HA) present in the iimbs of the treated animals is shown in Figure 9- Figure 9A shows 11 :u results for BMF2, BMPfc and BMP2/6 heterodirner, Figure SB shows the results. lor BMP;?, BMPG, BMPA and RMPF. For each of the designer BMPs, ectopic bone wais fphned ai doses at which wild type 8MP2 was unable in term a deteUabte bone mass, tn a head4e4lbsd comparison, at wild type 8MP2 with d&amp;ssgner BMPE was able to Induce ectopic bone to the same extent as void type 8MP2.S heterodimer, closely matching thu results obtained in the sn vitro experiments disclosed previously. Designer BMPs BMPG, 8MPA. and BfvlPF also demonstrated significantly higher ectopic bone formation compared to wild type BMP2 (Figure SB), Results from this assayisre shows in Figure S and else presented in Table 10, TABLE 10

5 BMP Receptor Binding

To further elucidate the mechanism of increased::fea^o^nfe activity: of the designer BiUSPs*. binding Kinetic analysis of each of the designer BMPs- witfl .a· pa$jH?|;--pfpjpM^ using the Octet system fRoiieBkT Monks Park, CA), The Octet QK analysis was performed at degrees in TBS with 0,1% TweerK2Q. Samples were agitated at ISOS rptn. Ahibhitenah igiu Octet tips were saturated with 10 ug/mi. of each meeptord>umah4g01-i% fusion protein for 20mio. which typfcaiiy respited in capture Saveis of the receptor that are saturated witii a row of eight tips. Each SMF was prepared as a sevenfold: serial dilution (typically 200-¾ pM in singilcate) plus buffer blanks, Each Repeptpr/8MP binding pair was run at least in duplicate Association was roohSgrepfor 10 min anti dissociation was followed fdr: 30 into buffer alone. Kinetic parameters (kon and calculated using the Octet

Data Analysis software 6.0 using a partial binding 1:1 modal fplipwihgmanufacturer's snstruotions.

The data.set forth in Table s? show, tfrat wild type BMB2 and BMP6 proteins, each demonstrated the expected high efhmb binding to woe i (ALK-3 and ALK-6; and type li receptors (ActRiiA. AclRIlB, and BMPR1I). respectively The wiki type 8MR2fb heterodkrmr exhibited high affinity binding to both groups; of type i and type ii receptors, as did: designer BMPG, which the type tl binding deewans A and 6 of SMP2 have been repiaced by the domains of wiid type 8MP8, Designer BfvtPE showed similar affinity as wild type BMP? for the type it receptors; as expected since no mutations were made in the type SI binding regions. Unexpectedly. designer 8MPE maintained high affinily binding for the type i receptors ALK-3 and ALK-S with t,he type i binding domain of SlvtP8 which has been subslituted In piac<2 for that of 813¾ whife atSP anaxpectedly bindsng the ALK-2 with a KD of 2 nm. Thus, 8MPE surprisingly gained a very high affinity tending for AIR-2 not observed In either WT BMP2 or WT BMP6, TABLE 12

13. combining tie mpfstioosof 8MPG and BMPE, or arginine at amino add residue 36 (P36R) redative to the amino acid sequence of |i#d type BMPS: as sot forth in SEQ ID NO;1< to produce BMF-GEP (also referred to as BMPGE Ρ36) and BMF-GER (also referred to as BMPQP P36R). respectively. produced designor BMPs which demonstrated high affinity, low hMKDs, lor alt typ# I and T ype tl BMP roooptdrs tnciudmg ALfGB, TA8BE13

Thus. the data disclosed herein demonstrate novel desi^^-:SW?s* .06$ URti^' &amp;

BiviP-GER arid 8MP-OEP. which combine the attributes of BMP-G and BMP-E sdch that these hovel designer BMPs demonstrate high affinity binding to 2 wMe repertoire el both type i and type ii weeptots. including. but not limited to, AIK2. ALKS, ALK6. AciRiiA, AotRiiB and BMPRiiA. The data further demonstrated that replacing the preliue at tesidue number 3¾ of the amino acid sequence of WT 8MR2 (SEO. ID NQ:1) io arginine produced a desig»ter BMP that was as odectiye as m otherwise identical BMP where the amino acid was not replaced. These novel osteogenic BMPs as atjempilflOd by 8MP-*3BR, provide Ngh levels ol biologic activity thus allowing lower dosing add. in some cases, mors rapid osteogenic response, strongly suggestion that these mot-sculee would provide highly effective therapeutics EMiAiptEA mtfumah primates

To: further assess the pofentiaf iherapepSd pofeotiai of the novel designer BMPs of the invention, 1j^iap#it$r af ·=3£%^:δ$$Ε #srot^par©d· &amp;'%$*' # - ^idliype BMP? in an rhr giorp human primate) fibula osteotomy model. A mld-dmphyeea: osteotomy of th;a fibula was 000000so b:iatefai|y wito the 1-mm blade of or: oscoaftog saw is; adult male Cynomoigus monkeys pVfacaea tesc&amp;U&amp;a} with a mean body we wist -and swnoard devtal-αη} of ? 5 t 0 ? kg and an ago range of seven to ton yearv A small inlramerinltory Kirschner wife was added to the previously described i-bular osteotomy model to main mis: aPpr-mont ot toe prawnra: and rile;a; bone end -, for more uniform torsional biomechanical lasting. The two major advantages of this modes ore the ability to us:1:sc a bilateral study design as a maul; of tho low morbidity of the procedure and tea ability la remove a 6 fa 8~am segment of the fibula containing she osteotomy site ?Of subsequent bicrneehowco: and histological evaluation without havmg to sacrifice toe emmai. Λ 500 w. solution of 0 5 mo/ml of either w<:d type or designer BMP was added a 30mm A 1 amm ACS sponge the sponge was wrapped around tne defect following surgery An approximately 2 mm I fuss Use of the ftouia of each lirrsb of a akelerally mature MNP was wrapped In an ACS sponge composing either a designer BMP m-aleoew at 0,5 sugar:! dose :250 pg total delivered} or inn same amount of wild typo BMP 2 m the contraUAemi hmb Thus each ammei iecewed wto type BMP In one tomb and a designer BMP to Ihs? contralateral iimfx

Ins thss made:, designer BMPB and BMPG were chosen since each represents a different class of designer molecule, designer BMPG shows high aihmty tof bod: type I and type II receptors while BMPh hinds the typo s receptor A: Kb? with high affinity In addition to binding type : receptors At K-3 end At K-G wild nsgh affinity. Radiographs were obtained every 2 weeks to compute the healing e>i the limbs treated with -ha designer BMP m«Reels camp-arsd with toe healing of the contralateral kmb treated wilt's «.«id lype BMP'2 :rs each ammal. Ac shewn :n Figures 1DA-10Q. the data, which «delude seven animals from eaels group, dernonsfrafed tost toe callus formed earlier and snore robustly in the limbs- if&amp;aisd with each dpsgtner BMP ;BMPE shown in Figure 10A and BMPG showr: in Figure 108-1 DC·} molecule compared to that with bone formation observed In the limb treated vein wiki type BRIP2.

Tables 14 rind 15, peiow. set.lorth data providing quantitative assessments of -he dsidrenee in bone mass and bone volume observed between limbs treated with wild type BMP2 and limits- treated with designer BMPF. As showr: sn figure 11. 8MPE administration resulted ;n an average of a 33% increase in bone volume {mm'') when compared with bone volume increase in wish type 8MP2 treated limbs. This pCT analysis included all the native bone whore there was callus* accordingly, BMP-E: was much more robust than BMP-2 in the same animals. TABLE' Μ Bone Maes <roa HAi

TABLE 15 Bone volume (mm 3}

Pro! me at position 36 rotative to the amino acid saquence of wild type BMPG as set forth in SFO IQ NOB 5? purportedly important in conferring Noggin resistance and providing increased ostcogen'ie activity !o wild type BMP? (see s,g . WG 3 DOS/OSGlot) Therefore, to asseos the sheet, of replacing P3G with a tx>n-eos rserved ammo acid substitution on foe novel activity of BfvIPGF, P3C of 8MPGBP was mtiiafod to argininsns to prodtice RMPGPP and osteogenic activity of the two designer molecules was assessed in vitro. 1 ha data disclosed herein so Figure 12 demonstrate that replacing P3G wtih arginine P35R) did not affect the binding affinity of the novel BMP-GE designer BMPs and both BGPGEP and BMPGER were as active as 8MP2;6 heterodimer.

As shown in figures 13 and '54, cat ectopic experiments show that BMP-OCR is as potent as BP1PPB&amp; al driving the formation of ectopic hone at the very loss dose of 0.25 ug total BMP when all molecules ere delivered on an ACS sponge Figure 13 shows that only 8MP-2/6 and BMP-GER. but not BMPE or BMPG. were significantly snore active ihan BMP-2 at th«s iow dose When the milligrams of HA termed in the ectopic ware quantities by m'TT analysis

The same samples were demineralized and scored tor bone fermatson iBone Score) by histology and these respite are shown in Figure -m. By tins method of scoring, at the low dose of 0.25ug delivered BMP-2 has no hone formation, and 8MP-GER and 2/6 had the highest score. 8MP-G and SfvIP-E were also significantly more potent than BMP- 2 but not as active m BMPA3ER: BMP-? <n in

Figures 15 and 16 show the results at a severe NMP titans osteotomy model comparing the activity of BMP-2 and RMP-GER. In this m«Oe: a wedge with and approximate width of 4-6 mm was. removed iot each fibula of trio NHP and pot Pack m place and held With a titanium pin The defect was then wrapped with an ACS sponge containing ?Gfi ug total BMP at a dose of O.Smgtoij In each NHP BMP-2 was placed in one Ismo and BMP-fitH was placed In ihe cOhfFaiaieraS limb, figure 1SA shows photographs of radiographs fatten at 5 waetts. showing the tielect In 4 of IP* 6 animals. The BMP-GEP «mbs showed significantly mom robust bone formation then those with BMP-2 Figure IbB (bottom panel of the figure) shows pCT images of the fibulas of the same 4 animafe following their sacrifice at week 10. As <on be seer, tic am jit o| bn; - n; u< ο ^ mvU mem iobusv in tho BMP-GLR «mbs than in the contralateral limbs treated with BMP2

Figure ιδΑ-G shows tho : analysis of these limbs -oaRlpaififiS' bone; volume compering the BMP-2 and 8MP-GER treated limb* from each animal. On average the 8MP-GER tnsafbd limbs raqusred 21% more torque to break fFigure 16A), were 24% more stiff (Figure 168), and the calluses were on average 56% iarger (Figure 16C) than the contiva iaferal BMP^; Ifdatod iimb. All of a-p yaiaia¥l^ than ,01 by pairwise Analysts; iTtese dftoshow lhef BMP-GER induced fracture repair ahd;t8i^;;fi^afio»::saQn8fc^^y abdtnore mbbstiy than BMP-2 in the same animal.

To further assess ihe effectiveness of BMPE bone foimafton in NHP. the ability of BMPE to induce osteogenesis in a wedge defect assay was compared to that of BMP2. Figure 17A-C shows radiographs of toe borsa fd^afibn defect model in three nori-homan pnmsfes vvbefe :t:Smg/mi of BMPAf was used: in one iimb end only 0,6 mg/mf gf BMP-GER wa used fi die ofierlMb using a calcium phbsphal® cement based carrier. Radiogfaphicaliy. the healingand bone fomtsfiph were equivalent. for each of the animals whether the freafment was with the high dose of BMP-2 or the lower dose of BMPdgERv Thbai;®yen at One-third the dose, BMPE was equivalent to BMP2 Ih indttoing bone formation, demonstrating the greatly increased activity of this designer BMP compared wifi wild type 8ΜΡ2. examples

CiysMlSizeiian BMP-2 and BMP-6

PijrsfsedVMI^gpmylatssy Wild type B^pa/i f^rodlmsr, MW type BfvSP2/2 homodimsc and wild type 8MP8/d horhodimer, each produced in mammalian bells, were concentrated to 5-10 mg/mi in fOmM sodium achats (pH 3.5), and crystallization attempts wars peilbmisd using a 'mosquito'' automated robotic setup at 18¾ (TTF Lablech inc,< Cauthudg®, ΜΑ), for each dimer and foe conditions were subsequently optimized to acquire crystals of good diffraction quality.

Crystals of 'Wild type BMP2/&amp; 8MP2/2 aifo 8K1P6/S were transiently styogfoteoiad add frozen in liquid nitrogen prior fo X-ray diffraction data eeliectlon at the synchrotron sources (ID beamtne of Advanced Photon Source SER-CAT), Data were processed and scaled using programs Mostim/Scala to deduce correct crystal tatte type and lo integrate/scaie data. The resdldtlon and unit ceil parameters are fisted as follows;: 8MP2/6 belongs to the space group of PdpXf with two copies of foe heterodfmer per , asymmetric unit- 4 <WfqM;ted-io 2 8A in one direction end 30A in the other two, with a unit ceil of a~b~ 1QS.28A. c=dS8.??A α~β~ν~90,:. BMP2/2 belongs to foe space group of P3r wifo two copies of the homodlmer oar navrnmetnc unit; it diffmcted to 2.7A with a unit cell ef a-fo-dfoTAfo rwlKBSOA, o~p~g(r'% y~ 120.' ΒΡΙΡδ’δ belongs to foe space group of P3,21 with one copy oi ghcb^SOdiiti^ M^metne·' unit; if diffracted to 2 6A with a unit ceil id a=h::S?.4QA. c~8S.0aA o'vp-SCh, γ~120\ Due ίο anisotropic diffracting nature of ΒΙΒι-'2.'6 crystals, foe data was e&amp;psofdaliy tru-icafbd and anisotfopicaliy scaled to preserve contribution of high-resolution dais

The structures of CHO BMP2/6. BMP2/2 and ΒΙνΙΡδ/δ were determined by molecular replacement method With program Phaser, using £ coh BMP2 iPDB accession: IREW; and £. coa 8MP6 (PDB accession: 2R52) as search models, After correct molecular replacement solutions were obtained and space groups confirmed* Phaser-caScuiafed electron dertsity maps were used to evaluate the quality of foe search nyddi4fo and fagiorfo in question (especially areas invobdhg type I and type ft feoeptot bSndipgf were stripped from foe original model forrebniiding in order to avoid model bias.

The simefuml models went through ugid-body refinement, followed by simulated annealing, ffositsonai and temperature factor refmemenl. Stripped areas were fbbullt using omit maps, and fob processes were repeated along with TtS :eiioerosnf unit! the nefthemaht stablfze<.l. the hnai refinement statistics are as follows: For SMP2/6. Rw/Rt ~ 0,2231 /0.2775. rmsd bonds ” 0.008 rrnsd angles s t S4S: For BMP2/2. Rw/Rt - 0 2114/0 20¾. rrnsd bonds - 0.00b, rmsd angles - 0,802: Per 8foPB/8 Rw/Rf -0.2170/0,2510. nusd bonds ~ 0,000, rmsd ahgfos A i f 0y M force straOfores are in; very good geometry based an Pmehec.k results.

The CHO BMP2/6 crystal structure t :.Jh ijSaftit^feifi f loop of CHO-prodnced BMP2 which is an impdi^ntfoihding mOllifor type !, feceptprs. Is differ^ *

corresponding region of E cols-produced and Irefokled BMP2, Jft the CiBO 8MP2 !oq> has a uniquely loopy” conformation when BMP?. which is mot's helical (Keller el ah, Nat Struct Moi Bid 11:481-486 {2004)) The data demonstrated that the 053 of CHO-produced BMP2 points towards the rctoeptw'interface, while the H54 points; away from the receptor as shown in Figure 3A. in E: =ροίfits away from the

Moepter and the H54 lines up toward the receptor (retefred id heMn as a djistidine deorstop”), stacking against a proisne residue (P4S) on the 8fvlP2 type t receptor Aik3 m shown in Figure 3B (H54 is aiternatiyelydaheied H336) Without wishing to be bound bv any particular theory tills stacking cotM prevent the type I receptor tram fully' binding la F coii refolded RMP2. explaining the reduced binding activity el L col I BMP2 when compared w*th CHQ BMP2. This structural feature is illustrated in Figure 3A-8. in this figure, histidine 64 {H54) is numbered as H336. asparagine 56 (NS8) is labeled B33B, and pu:j of ALK3 is shown in darker gray.

As illustrated In Figure 4, fully glycosylated OHO BfvIFB also has this 'doorstop'’ histidine residue pc!riling Imo the receptor binding site This doorstop His structural motsf is a common structural ionium among BMPs {excluding CHO BtvtP2) (see e,g., Keiler el ai.., Nat SUuci Mo! 3;ol 11:481-8 {2004V Katzsch si a/, PM BO J 28:937-47 (2009) Without wishing to be bound by any particular theory, It may be that a specific glycan of CHQ BfdP2 is linked though extensive hydrogen bonding with arginine 16 fihs giyean tether'' also designated as 11296) This stye* tether is insfrated in Figure 4A and its Interaction with bie gfpan is depicted using dotted imes between the gfydan and fits tether R293 Which ia aiso referred to herein as R16. Thus, without wishing to be bmtod by eny pjantoujar fhegryyihe glypan tothpi may serve to stshiiise the conformation of the pro-hoiicai loop of the BMP2 molecule such thsifto: histidine doarstopvif otherwise present, is instead oriented away from the type i receptor interface thereby aiioWing the ligand to contact the receptor to a greater extant than sn the presence of the histidine doorstop. In ether words, the re-orientation of the histidine doorstop as observed in CHO BMF^? is most My to bd the consequence of giycan tethering. -W$^t &amp;*Mhd by any; patibular theory) the data disciosed herein suggest that where the histidine doorstop is present, removal of the doorstop in the: absence of glycssytetlon by introducing a mutton that changes ite orientation of the His away increases: binding of the BMP ligand with the type I receptor.

Designer li^PE wfdet? contains a Sow affinity type if binding domain # BfilP2 and a low affinity type i binding domain similar to that of 8MP6, shows (f} Increased osteogenic activity in both In vitro and inwiio assays; and (2) has an unexpected gain of function to bind AfK£ a type i receptor, despite the presence of a iow affinity type f receptor binding domain. Without Wishing hound by any pafilcufar theory, it may be fivu inis surprising discovery is mediated by ntoiflpie hydrogen between

Mis giycan morsCes and ine Hi6 fihe "giycan tether") m the type i receptor-binding domain of BhtPE, Thfe tefhenng Interaction may mediate a structumi rearrangement at the pre-hailcai region of the BMPF motet uto that presents a proper binding surface for Aik2 by positing H54 (the ‘doorstop'') sway from the interface thereby avowing closer interaction between the BMP and the receptor in contrast, asJiuslraied in Rgufe 4Β, 8ΜΡ6 which also has a low aifindy type i binding domain similar (¾ that of BMPE. does not bind Aik2 because its 'giycan tether' {R413; which wouM Da needed to tether ds giycan moieties. is shifted :d icca-ion when compered to the BMPE tether ·Ρ?^νΒΡ6'. Toes, ,n BN f 6, the ilywm as not tethered and the doorstop (H454) so not positioned away iron? the ligand-receptor interface, i bo '‘giycan tether” appears to be a phenomenon unique to wild type giyco&amp;yiated BMP2 (as exemplified by 8MP2 produced in CHO ceils), and structural remodeling of the prsheiseal loop of BMPs by si Producing (or removing- 'gives n iwinef" can new bn used, for the first tens, in modulate Sype I receptor-binding ability of other LiMPs. therefore. one skilieu m the art. now armed with the teachings provided hero in, would understand how to mutate the BMP in order to position the doCiSlop away from id* rapeptor inmrface by introducing mutations that swing the 1154 away or by affecting the glvoap Mfldr sb that teihemig medial®* the shift -a Hfyi and wouid further appreciate that these teachings can be used to design a BMP with increased {ordecraasad rf mutations ore introduced to swing H54 into fh® ddprstbp position; binding to Its receptor or to create designer BMPs with galn-of-lunction mutations such that they bind to hovel receptors that they did previously bmd As more fully set forth below, the; present Invention demonsiratss new to use this novel doorstopftether cawgn method to design improved' osteogenic proteins. Thus, the present invention provides a novel -method lor misonai dosage of iroproyad oslbogenio proteins comprising altered receptor binding, to orde t < moir ini \ imdemtoi'd w>' it Tk* =¾ 4¾¾ BMP-€ and BMP-GBR binding to AlK·?, and to further elucidate this novel mechanism cl affecting receptor binding using the doorstop/giyean Miter, the cryeta· structure of SMP-E was solved and compared to that of 8MP-2 and 8MP-8. The hey structural findings are shown in Figures 16 and 19. As illustrated In Figure IB BMP-E maintains the ordered sugar of BMP-? while maintaining the centra! heiica! structure of BMP“-S. The structure shown in Figure 18 demonstrates that BMF-F, and presumably BMP-OER, Is dliferoni from both BMP-2 and BMP-6 in the critical region of type \ receptor binding. Figure 19 is a blow-up empaling the area surrounding the potential His doorstep ot 8MFE (light gray) and SMPb fdarfe gray)-5 "IN· '#lpam· the simifarity of the alignment of the histidine and asparagine in both molecuiaa and also .shews the difference in give an positioning and demonsiratsrig the tethering of the BMPE giycan by Ri&amp; (the tether) which also causes a mom rigid conformation oi the giycan such that a longer giycan Is rendered: for BMpp by the analysis compered wiif: die slimier giycan rendered for BMPb (In dark gmyi in order to determine if the giycan ot 8MF-E is driving the interaction! with and Its higher activity BMP-'T BMPAi, and BMF-B were treated with Endo H To clip the |s;twb ^cNae UpllSy

The bfi#hg.: :Ρί·^Ρτ£·Ι^' AIK-2 decrsassid to #0 nM whereas ft’s ahinity for ALK-3 and ALK-6 were sti ih the 3¾ nM radge shewing the intact carbohydrate fa ekbemeiy important for thi® interaction. The acidly of this degiptapted metont aiso deoroas^ algniftdahtly. As shown In Figure 20-,. in this espenmerii the Ehdo H treated degiycosyiated BMP-E activity shifts to the right and is aimost. equiyaienf to BMP-δ WT fhe LC-50 shiits from 3nM to approximately 50 fiM, These data show that the carbohydrate of BMP-F is essential for its. activity, and this should it has the same regionofinto BMP-2 with only toe ftger domains ditto ring, Since; too carbohydrate is osseniM far increased: recapiaf binding and osteogenic activity, these resuits indicate indicates that production of BMP-E or BMP-C>rR in E. coli, or any other system lacking giycosylatom Would not produce a BMP with activity superior to BMP-2 Wf

Purified, toily-glycosyiated BfvfF^E, was conoeniratud to 8 ? mg'infto SSiyfM sodium; dedtete <pH 3 5), and crystallization attempts were performed using a “mosquito' automated roihOilO setup at 1ΑΌ (ΠΡ LabTeeh lnc.; Cambndyo. MA). initial crystallization bibs were obtained for seen dimer ano too conditions ware subsequently optimized to acquire crystals of pood dsffr&amp;ciipo quality.

Crystals of Uf#--£ wvte transiently cryoprotsctou fjfozert.liquid rliroph prior to X^ray: diffraction data collection at the synchrotron sources {ID beamiln* of Advanced Photon Source 3ER-CAT}. Data were processed and scaled using programs MosflnvScaia in the CCP4 psokag© to deduce correct crystal lattice type and to integrate/scaia data, Die tosoiutlon and unit osil paiamoiem ^re listed as follows- BMPB belongs to the space group of 94-2,2 with two Espies of foe BpBE id toaeft esymhtotob unit; it diftraofed to 2JA. with a unit cell of a~b-5?.?8A, c~14>S,0lA.t o«S~y”S30,

Tbs structure ot BMP£ was determined by using fully glycosylated CHO BMP2 and BMFft, deletftjifnsd at Riser as search rdodeis. After correct moiocoiar repiac-ement solutions were t Pbas^pftidiilated electron density maps were used to evaluate the quaifty of tbd: aeatdb btodels, arid regions; sn qud^idn (especially arses around type 1 receptor binding and giycdsylaiion} were stripped from the original model for rebuilding In order to avoid model bias.

The structural model ot BMPE went through rigid-body refinement, followed by simulated annealing, positional and tumpomtusv factor refinement using program Phenix, Stripped areas were rebuilt using omit maps, and the processes were repeated along with TLA refinement until the refinement stabilized. The final refinement statistics are. Rw Rf - 0,2252/0.2840, rmsd bonds - 0 008, rmsd.angles -0.938, The structure is in very good geometry based on Procheck results, BViPE a designer molecule wherein residues 44-80 of BiVPto replaced by the corresponding region from BMPS, maintains the overall framework of BMP2 while possessing the Type I receptor-binding segment of BMPS As shown m Figure 2i toe crystal structure revealed that foe grafted segment; still retains a similar conformation as in BIVlFft, forming a small helix in foe pre~hci;cal loop Within which * ihe "·dp«^op*' NB4 :Nowever. witopift wishing to be bound by^«y i^^aa{«if·;··. theory, ft may be ifoa£diieffofoe:p5^^-;df:%fyc^.tethi^e^ at Ri ft and El 10* |E108 of BMP -21, both of Which fotrn multiple :tfrird: 'a;ri# (ft-mannose and «-mannose, respectively}, the extended giyebaylatiGn chain Is attaohed: to the protein; surface; exactly as seen In CH0 BMP2 The tethering of gfyean chain also dislocated the altout 2Λ |i referehca to the overall Iramewcrt·;. Without wishing to be bound by any particular theory, if may be'fe:'|ip:i^jrp«1isiii%' discovery ihaf the StvIPfrilke pre-hebca: loop combined with th® "BMP2-iiks giyimsyktooh prssenf a bindmg epitope tor toe A&amp;2 receptor, which does not normally sntersct with either BMP2 'dr BftXPfh Degivcosyiation renders BMPE sncapabJa of binding to Atk2, whichnndbfshbfesthe iihp^rtsnbe of giyco&amp;ylation in mediating Aik.2 recognition tor BMPE. EXAMPi. F 6

Nmitmn R&amp;sHsisrtca in order to investigate if resistance to the secreted BMP inhibitor Noggin would increase the activity of BMP-GicR or 8MP-E. these pofertfiai toet^eutk- molecules Were further mod-bed to potentially increase the:r resistance to Noggin Recently. it wan demonstrated that m E. coil-p-reduced proteins incorporation or a Oterminal portion oi activin-A into wild typo BMP2 increased resistance to Noggin Inhibition, See WO 2010/0392IB at. e o.. Figures IS and 16, Theretore to determine whether the nevei designer proteins disclosed herein could he improved even further- fey incorporation # atdMreA sequences, the Noggin resistance (NR) ammo acid sequences were substituted Into IBMP-E (SEQ I Q NO: 12) and BMP GER {SEQ ID NO:3?) to produce BMP-&amp;NR (SEQ SDNO:®) and BMP-GER-NR (AEG ID NQ:?'t). As shown in Figure 22 BMP-E.-NR and BNIP-GER-NR have equivaisnt in vitro activity in an Alkaline phosphate activity assay compared wuh Sh#€' and BMP-GER and are cdtopieii#y feslstoht M Noggin while 8MP-E and BMP-GEE are sensitive to Noogm

To understand the potential baste to: the Noggin resistance demonstrated in viSto fey 6MP€>UR and 8MP-GLK-NR. the binding affinity oi these molecules for % typo {{ -&amp;«Μ.β·. 'ms- assessed. As shown in Table 16. below. aefivin-A is unable to bind Noggin but: the Noggin resistant SRIP-E,NR and: BMP-GER-NR bind Noggin, but not as strongty as BMP-2. BMP-E. or BNIP-GER Those data also show that the Noggin resistant BMPs bind the type it BMP receptor ActRilB with extremely high affinity mat is even higher than that of BMP-GER. Without wishing to be bound fey any partiehidf theory,: these data suggests mat BMP-GER-NR and BMP-E-NR are resistant to Noggin due to thee ntuoh higher : affinity lor the BMP type II receptors than that of Noggin and are therefore able to bind even in the presence of high amounts of Noggin.

TABLETS

Aiougb the 8MP--E and oMR-GER molecules cosnprisittg the Noggin remstaht portions of Noggin resistance in vitro, mess mauils did not correlate to improved in vivo betivify.; Tiiai ls. ^'hort the osteogenic activity of these BMP^NR and BMF-GER*NR was compared with that Of iiMtFC'ond BMP-GFR in a rat ectopic assay, the NR molecules were mych less pofenl This date if shown in Figures 23 and 24, Mete speeiilcaiiy. the Bone Scots for BMPuGER and BMP--GER-NR was compared and, at alt concentrations tested {0.13¾ yg, 0.5¾ yg, 0.S pg. and 1,0 yg}; BMP-GER greatly ootpehtymed BMP'GER NR as shown in Figure 23 Similarly:, Figure 24 demonsMes that BMP-E; produced a much: higher Bone Score; compared evith BMP-E~NR in this in vivo assay,. Thud,;; for hath BMP-E and BMpvGER tne purportedly ;Noggin resistant versions wbre much iess pptdnf in vivo than their NR {Noggin resistant) counterparts. and in the case of BMPdT aimosi ait in vivo activity was tost due to incorporation of sequences of achvin-A {see Figure 24 comparing

These data demonstrate that incorporation of sequences potentially conferring Noggin resistance.. While increasing binding for certain type is mceptom fag . ActRdo}, did not increase in vivo osteogdhic activity of the designer BMP.

Further although the addition of Noggin did not improve the osteogenic activity of ?hd designer BMPs in vivo, indeed, si appeared to decrease thesr in vivo activity, the novel designer BMPs of the inventinn demonstrate greatly increased coseogenic characteristics. compared with wild type BMP and provide potential nove; therapeutics lor a wide vaneiy of applications evnn without defnenshahng Noggin resistance in vitro. Theratore, the designer BMPs of the inv&amp;mion provide remarkable qovei potential therapeuVs demonstrating a gr&amp;sfv >mproved dmsca! prohie for. among other uses, hope augmentation and; repair,

Thp dididosums of eacirarid every patent, patent application, and pubNcai»on cited herein are hereby incorporated herein by reference in their entirety.

While the invention has been disclosed wrth rnferenog to apepffih embc^inienls, if is epparepf Stht omer embodimerits and variations* of this invention ragy 'p^^fsktfted inThe aft- without; departirig bom the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims (17)

1. A designer BMP protein comprising: a first set of mutations with respect to SEQ. ID NO:1 comprising V33I, P36R, H39A, H44D, P48S, A52N, D53A, L55M, S57A, N68H, S69L, V70M, insertion of a P after N71, S72E, K73Y, I74V, A77P, and V80A, wherein the designer BMP protein exhibits altered binding to each of a type I and a type II BMP receptor compared with the binding to the type I or type II receptor by a corresponding wild-type BMP.

2. The designer BMP protein of claim 1, further comprising: a second set of mutations with respect to SEQ. ID NO:1, comprising E83K, S85R, A86P, I87M, L92Y, E94D, N95G, E96Q, K97N, V98I, V99I, L100K, N102D, Y1031, D105N, V107I, G110E, and R114S.

3. The designer BMP protein of claim 2, wherein the protein binds an ALK2 receptor with a KD not greater than about 2 nM.

4. The designer BMP protein of claim 1, wherein the protein binds an ALK2 receptor with a KD not greater than about 2 nM.

5. The designer BMP protein of claim 1, wherein the protein binds each of an ALK3 receptor and an ActRIIA receptor with a KD not greater than about 2 nM.

6. The designer BMP protein of claim 1, wherein the protein binds each of an ALK2 receptor, an ALK3 receptor, an ALK6 receptor, an ActRIIA receptor, an ActRIIB receptor, and a BMPRIIA receptor, each with a KD not greater than about 10 nM.

7. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the designer BMP protein of claim 1.

8. A designer BMP protein comprising the following mutations with respect to SEQ. ID NO:1: V33I, P36R, H39A, H44D, P48S, A52N, D53A, L55M, S57A, N68H, S69L, V70M, insertion of a P after N71, S72E, K73Y, I74V, A77P, V80A, E83K, S85R, A86P, I87M, L92Y, E94D, N95G, E96Q, K97N, V98I, V99I, L100K, N102D, Y103I, D105N, V107I, G110E, and R114S, wherein the designer BMP protein exhibits altered binding to each of a type I and a type II BMP receptor compared with the binding to the type I or type II receptor by a corresponding wild-type BMP.

9. The designer BMP protein of claim 8 having an affinity for the ActRllb receptor of less than about 1 nM.

10. The designer BMP protein of claim 8 having a Kon with respect to the ActRllb receptor of about 1x106.

11. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the designer BMP protein of claim 8.

12. A designer BMP protein comprising the following mutations with respect to SEQ. ID NO:1: V33I, P36R, H39A, H44D, P48S, A52N, D53A, L55M, S57A, N68H, S69L, V70M, insertion of a P after N71, S72E, K73Y, I74V, A77P, V80A, E83K, S85R, A86P, I87M, L92Y, E94D, N95G, E96Q, K97N, V98I, V99I, L100K, N102D, Y103I, D105N, V107I, G110E, and R114S, having an affinity for the ActRllb receptor of less than 1 nM.

13. The designer BMP protein of claim 12 having a Kon with respect to the ActRllb receptor of about 1x106.

14. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the designer BMP protein of claim 12.

15. The designer BMP protein of claim 12, wherein the protein binds an ALK2 receptor with a KD not greater than about 2 nM.

16. The designer BMP protein of claim 12, wherein the protein binds each of an ALK3 receptor and an ActRIIA receptor with a KD not greater than about 2 nM.

17. The designer BMP protein of claim 12, wherein the protein binds each of an ALK2 receptor, an ALK3 receptor, an ALK6 receptor, an ActRIIA receptor, an ActRIIB receptor, and a BMPRIIA receptor, each with a KD not greater than about 10 nM.