FI119373B - Bone proteins - Google Patents

Description

119373

FIELD OF THE INVENTION Field of the Invention This invention relates to bone formation inducing proteins called bone morphogenetic proteins (BMPs), particularly BMP-4; nucleic acid molecules encoding said proteins; vectors containing said nucleic acid molecules; and host cells expressing said protein. The present invention also relates to the use of these bone morphogenetic proteins in the treatment of disorders such as those associated with bone and cartilage formation. The present invention further relates to osteogenic devices and pharmaceutical compositions containing said proteins.

BACKGROUND OF THE INVENTION

In 1945, Lancroix discovered a phenomenon related to bone formation when he showed that acidic alcohol-bone extract induces heterotropic bone formation at ectopic sites. Twenty years later, Urist and his colleagues decalcified the bone matrix, and when it was implanted into muscle, he noticed new cartilage and bone forming at the implant site. These findings led to the isolation and: V: purification of a bone inducing agent, designated BMP, from bone matrices of various animal species and, years later, of these new. cloning and characterization of cDNA for proteins. BMP biological activity · has been determined by bioassay in BMP implanted into rat or mouse muscle muscle or in mammalian cell cultures measuring alkaline phosphatase: * Y (ALP).

Earlier studies since 1965 have shown that BMPs belong to the TGF-β superfamily, and like other members of this gene family: .Y 30 multiple effects on cell motility, growth, and differentiation, particularly bone formation and tissue repair processes:. but also in embryogenesis and cancer. They are small molecular size, hydrophobic glycoproteins that are soluble in chaotropic agents such as urea and guanidine hydrochloride but are resistant to several proteases, such as collagenases.

BMPs are produced as large precursor molecules that are processed into proteolytically mature peptides after translation. Like other members of the TGF-β-2 119373 superfamily, BMPs contain a sequence of seven cysteine residues in the mature C-terminal portion. In mature BMP monomers, between these cysteines, there are three disulfide bonds and one disulfide bond that combines the two monomers to form a biologically active dimer.

The effect of BMP is mediated through specific receptors on the cell membrane of target cells. BMP receptors are serine-threonine kinases that are similar to TGF-? Receptors and are divided into two subgroups: type I and type II receptors. BMPs can bind tightly to only 10 receptors that form a heterotetrameric complex. The formation of such a complex is a prerequisite for BMP signal transduction. Within the target cell, BMP signals are transferred to the nucleus using specific molecules specialized in signal transduction called Smadit, which are also capable of attenuating BMP signals.

To date, 16 different BMP molecules have been characterized, seven of which (BMPs 2-7 and 9) have been shown to be able to induce bone formation when implanted into ectopic sites. Based on the amino acid sequence of the mature portion, the BMPs are divided into two subgroups. BMPs 2 and 4 are 86% identical to each other and BMPs 5, 6 and 7 are 78% identical. The identity between the two subgroups is only about 56%. The amino acid sequence of BMP-3 is approximately 45% similar to that of BMP-2 and BMP-4 and BMP-9 is 50-55%:: * · identical to BMPs 2, 4, 5, 6 and 7 with. Due to the high homology and small differences in size of the molecules, purification and identification of the BMPs at the protein level is quite difficult, time consuming and expensive. Therefore, most BMPs are produced today. utilizing the molecular biology methods of * · · \\ V. Various recombinant protein techniques have been tried, and both eukaryotic and prokaryotic systems have been used.

30 Most of the research has focused on recombinant human BMP, but the Cervidae is also an interesting area of study because of: efficient bone induction in horns. The horns are bone-bearing skull. ···. ' structures typical of the Ce / v / dae family, which differ from the Bovidae • · horns due to their growth pattern. Horns grow from the tip of the horn and males: * ·· 35 drop them once a year. It has been claimed that horns are the fastest growing structures in mammals and these structures are known to be the only ones that renew completely every year. A certain type of endochondral ossification occurs during the formation of horns, in which the process proceeds through 3,119,373 rich vascular cartilage, which eventually becomes bone upon calcification. The horns provide an interesting model for the regeneration of mineralizing tissue in an adult animal, and bone regeneration continues until the horns are dropped. Although the root cause of the astonishing 5 growth rate of horns has not been elucidated, several hormone BMPs are known to exist in horns. Deer horns have been shown to produce BMP-2 and BMP-4 (Feng et al. 1997 Biochim Biophys Acta 1350: 47-52; Feng et al. 1995 Biochim Biophys Acta 1263: 163-168). In addition, BMP-3b is expressed in reindeer horns (Kapanen et al. 2002 J Biomed Mat Res 59: 78-83). However, it is also possible that the horns have one or more unknown factor (s) causing rapid horn growth.

Because of their osteoinductive properties, both BMPs extracted from demineralized bone matrix and BMPs produced using recombinant techniques are very interesting and highly potential alternatives for bone graft. Various BMP proteins have been used in several experimental and clinical studies.

Bone morphogenetic protein 4 has been isolated from various sources, including some mammalian species such as human, mouse, rat, rabbit and dog. Like BMP-2, it is also isolated from the Texas deer horn of the Cerv / dae family (Feng et al. 1997; Feng et al. 1995). However, the function and effects of the deer BMP-4 have not been demonstrated as it has not been produced by any expression system.

To date, BMP-3b is the only BMP that has been characterized by j * V in reindeer (Kapanen et al. 2002).

Several in vitro studies have shown that BMP-4 signaling is required in chondrogenesis to convert chondrocyte precursors to cartilage cells. Its expression is elevated by the fracture and, like BMP-3, -7, and -8, is expressed for a limited time from day 14 to day 21. It:! ·. expressed in street-forming tissue prior to call · · · V / formation. It also acts as a chemical attractant for human V primary mesenchymal stem cells. In addition, BMP-4 has been shown to play an important role in the induction of bone formation, both in vivo and in vitro, and, like "**: BMP-2, its effect on embryogenesis cannot be replaced by any other BMP.

Although the functions and activity of BMP-4 have been the subject of steady research for over ten years, and although human BMP-4 is produced as a recombinant protein, 119373 4, at least to date, has not provided data on the biological activity of BMP-4 cloned in horn tissue, from a member of the Ce / v / afae family.

US 6245889 discloses purified BMP-2 and BMP-4 proteins and methods for their production. Also disclosed is a pharmaceutical composition containing BMP-4. As is generally known in the art, these proteins and compositions may be used in the treatment of bone and cartilage defects, as well as in the treatment of wounds and the repair of similar other tissue lesions. In addition, the above pharmaceutical composition may include a matrix capable of delivering said BMP protein at the site of bone and / or cartilage injury and providing structure to the developing bone and cartilage, and also capable of being optimally resorbed by the body. Such matrices can be formed from materials currently in use in other medical implant applications.

US 5,399,677 discloses DNA molecules encoding mutant forms of bone morphogenetic proteins. Mutant forms of BMP can be produced in bacteria and folded into a biologically active form either as a homodimer or heterodimer of BMP. The method by which the mutant BMP in question is made is also shown. These mutant forms are useful because they are flawlessly folded when produced in a bacterial host.

• · • · · · · · ·; WO 98/51354 discloses osteogenic devices and methods of using them for the repair of bone and cartilage defects. A method for growing new bone at 25 lesioned bone sites in a mammal comprises the step of implanting a? · ·? V calcium phosphate matrix containing at least one osteogenic protein into the lesion site. These osteogenic proteins comprise a number of · * · * · * morphogens, such as bone morphogenetic proteins.

EP 1131087 discloses the additional use of morphogenetic proteins such as BMP. It has been shown that the contact of cancer cells with morphogens inhibits:. cancer cells and causes their differentiation away from the cancer cell. '•• V phenotype. The use of morphogens can affect the cellular fate of cancer cells and · · contribute to reducing the symptoms of cancer. BMP-4 belongs to the preferred morphogens • ·: * ·· 35.

• ·

Although the applications of some BMPs as inducers of bone and cartilage formation, as well as in the alleviation of the symptoms of cancer, are known, there is a need to obtain even better methods for isolating these proteins, than before. Such proteins would be of use in better therapeutic methods and applications.

Summary of the Invention

Surprisingly, the novel BMP-4 protein of the present invention, isolated from a reindeer and having high sequence homology to other already known bone morphogenetic proteins, has highly beneficial properties related to bone and cartilage formation. These properties are significantly better than those of the already known BMP proteins. The bone morphogenetic proteins of the present invention and their homologs are useful as bone and cartilage inducers in a variety of applications, such as therapeutic applications.

One aspect of the present invention pertains to an isolated morphogenetic protein, or homologue, analogue, derivative or fragment thereof, which contains the essential amino acids of the amino acid sequence of SEQ ID NO: 1.

20

Another aspect of this invention relates to an isolated DNA molecule encoding said morphogenetic protein.

Furthermore, another aspect of the present invention pertains to a nucleic acid vector which: *. ·! 25 contains said isolated DNA molecule.

In another aspect, the present invention relates to a recombinant host cell comprising said DNA molecule or the aforementioned nucleic acid vector.

A further aspect of this invention relates to a bone morphogenetic protein produced by growing said host cell so that it:. expresses said bone morphogenetic protein and said bone morphogenetic protein is recovered from said host cell.

Another aspect of this invention relates to a recombinant host cell, * · **: which expresses said bone morphogenetic protein.

119373 6

Yet another aspect of the present invention pertains to a pharmaceutical composition comprising said bone morphogenetic protein.

Still another aspect of the present invention relates to said isolated bone morphogenetic protein for use as a medicament.

Yet another aspect of the present invention pertains to the use of said isolated bone morphogenetic protein in the manufacture of a medicament for use in the treatment of bone and cartilage disorders where regeneration, repair or growth is desirable, or other diseases such as cancers.

Yet another aspect of the present invention pertains to an osteogenic device for treating said disorders, which device comprises a morphogenetic protein of said bone.

15

Yet another aspect of the present invention pertains to a method of inducing cartilage and / or bone formation by treating said cartilage and / or bone with said isolated bone morphogenetic protein.

Yet another aspect of the present invention pertains to a method of treating such bone and cartilage disorders where regeneration, repair, or growth is desirable, or other diseases such as cancer by administering said isolated bone morphogenetic protein to a patient suffering from said disease. . ·, * · Malfunction.

Brief Summary of the Figures Figure 1 shows plasmids containing rdBMP-4 inserts in the PCR vector pGEM- T® (Promega).

Figure 2 shows plasmids containing rdBMP-4 inserts in the expression vector: pTrcHis2A (Invitrogen).

• · ·

Ml I

Figure 1 shows plasmids containing rdBMP-4 inserts in the expression vector: · ··· 35 pET-22b (+) (Novagen).

♦ • ·

Figure 4 shows plasmids containing the rdBMP-4 insert in the expression vector p1VEX2.4c (Roche).

119373 7

Figure 5 shows the amino acid and nucleotide sequences of the mature portion of reindeer BMP-4 protein produced in the pTrcrd4 / 116 expression plasmid. The mature portion of reindeer BMP-4 protein is flanked. Cysteines that are typical of the TGF-β superfamily are indicated in bold.

5

Figure 6 shows the amino acid and nucleotide sequences of the mature portion of reindeer BMP-4 protein produced in the pETrd4 / 116 expression plasmid. The mature portion of reindeer BMP-4 protein is flanked. Cysteines typical of the TGF-β superfamily are indicated in bold.

10

Figure 7 shows the amino acid and nucleotide sequences of the mature portion of the reindeer BMP-4 recombinant protein produced in p1VEXrd4 / 116 expression plasmid. The mature portion of reindeer BMP-4 protein is flanked. Cysteines that are typical of the TGF-β superfamily are indicated in bold.

15

Figure 8 shows a Coomassie stained SDS-PAGE with fractions eluted from a HiTrap column (Example 3C). The zones represent 1) starting material; 2) material passed through the column; 3) first wash; 4) second wash; 5) the standard; 6) pH gradient elution, pH 6.2; 7) pH gradient elution pH 5.3; 8) pH-20 gradient elution, pH 4.0.

: Vi Figure 9 shows X-rays of the hind foot muscles. A) a foot implanted implant containing human BMP-4 protein and B) a foot implanted implant,

* ·· I

containing the BMP-4 protein of the present invention.

Detailed Description of the Invention · · · · 25

• I

***** The homology between the mature portions of the previously known BMP-4 proteins in mammals is very high, being 100% between mouse and rat. Comparing these two 30 mature parts with the Texas six deer and the corresponding mature parts ···, the homology is 99% (Table 1). Cloning of the mature part of reindeer BMP-4 and:. *. characterization showed that at amino acid level the highest homology is with Texas. ···] with six deer, 99%, and with 98% homology with mouse, rat human • ♦ I ”and canine BMP-4. At the amino acid level, the lowest homology to reindeer BMP-4: *** 35 is with the corresponding rabbit protein (96%). At the nucleic acid level, homology between reindeer and Texas deer BMP-4 reaches 99% and between reindeer and human 93%. The lowest homology at the nucleic acid level, 88%, was obtained by comparing 119373 with the 8 reindeer BMP-4 rat. In general, BMP-4 also has homology with other types of BMP, such as BMP-2.

Origin Reindeer Deer Human Mouse Rat Rabbit Dog _ nt ah nt ah nt ah nt ah nt ah nt ah nt ah

Poro 100 100 99 99 93 98 89 98 88 98 93 96 94 98

Peura 99 99 100 100 93 99 89 99 89 99 94 97 95 99

Human 93 98 93 99 100 100 91 99 89 99 94 94 95 99

Mouse__89 98 89 99 91 99 100 100 95 100 89 96 89 98

Rat 88 98 89 99 89 99 95 100 100 100 88 96 89 98

Rabbit 93 96 94 97 94 94 89 96 88 96 100 100 94 96

Dog__94 98 95 99 95 99 89 98 89 98 94 96 100 100 5 Table 1 Homology of the mature portions of BMP-4 in mammals at the nucleotide and amino acid levels (%) (nt = nucleotides; ah = amino acids).

The alignment below shows the amino acid sequences of the mature portion of human and reindeer BMP-4 proteins (comparison made using ClustalX 1.8 (Thompson, 10 JD, Higgins, DG and Gibson, TJ (1994) Nucleic Acids Research, 22: 4673-4680)), rdBMP -4 = reindeer BMP-4, hBMP-4 = human BMP-4, star indicates identical amino acids). These amino acid sequences differ by only two amino acids • · · · 1..1. for: amino acid 6 is serine in humans and proline in reindeer (Ser6 → Pro6), and T! amino acid 21 is leucine in humans and proline in reindeer (Leu21 → Pro21). Surprisingly, / / 15 difference of only two amino acids leads to major changes in the function and efficiency of these two BMP-4- • · · proteins. Because proline is a specific amino acid for J ·: because it generally has a major effect on protein structure and folding, it can be readily seen that the beneficial effects of this invention are related to these two amino acids. In particular, proline 21, which 20 replaces highly conserved leucine, may play an important role in this invention. The closest equivalent of reindeer BMP-4 is human BMP-4, which is ···.

• · · ··· · »· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · whether-and-bichard

rdBMP-4 S PKHHPQRARKKNKNCRRHS PYVDFSDVGWNDWIVAPPGYQAFYCHGDC PFPLADHLNST

hBMP-4 SPKHHSQRARKKNKNCRRHSLYVDFSDVGWNDWIVAPPGYQAFYCHGDCPFPLADHLNST

***** ^ ************** ****************************** *********

5 rdBMP-4 NHAIVQTLVNSVNS SIPKACCVPTELSAISMLYLDEYDKWLKNYQEMWEGCGCR

hBMP - 4 NHAIVQTLVNSVNS SI PKACCVPTELSAI SMLYLDEYDKWLKNYQEMWEGCGCR

******* + *****, ********* νΙί ************ ^ ****** ^ ****** **** · ** · * · 10 "BMP-4 protein of the invention" or "bone morphogenetic protein of the invention" refers to a protein that has bone morphogenetic activity (or morphogen, both of which may be used synonymously), such as BMP isolated from reindeer The 4 protein described herein (SEQ ID NO: 1 in the Sequence Listing or rdBMP-4 Alignment above), and includes homologues, analogs, derivatives, and fragments thereof. Such homologues or derivatives include functional derivatives of the protein of interest, such as proteins derived from the parent BMP-4 protein or any other BMP of any species. Derivatives may differ in their length and may include amino acid additions, deletions and substitutions as well known to those skilled in the art. A characteristic feature of the bone morphogenetic protein of the present invention is the two essential amino acids, amino acids 6 and / or 21, of the mature portion of the protein, both of which are proline as described above:. alignments. Preferably, the regions in which these amino acids are located are. '·] | preserved in the BMP protein of the present invention.

• · * 25 • · ·

On the other hand, additions, deletions, and substitutions far from said characteristic region are unlikely to cause a substantial change in the function, potency, or folding of the BMP protein of the invention. For example, homologues with deletions, such as deletion of a few amino acids, preferably 1-10: 30 amino acids, more preferably 1-5 amino acids, most preferably 1-3 amino acids, at either the carboxyl or amino terminus, wherein the polypeptide is: I *. shorter are within the scope of this invention so long as such deletions do not affect the characteristic amino acids of the BMP protein of the invention. It is preferred that the 7 homologues in question have the beneficial ··: * ·· 35 properties of the original reindeer BMP-4 protein associated with said characteristic amino acids Pro6 and / or *: **: Pro21 and / or the region around them. Said homologues may have amino acid substitutions that do not substantially affect the function and potency of said protein. For example, an amino acid not located at or near active 119373 position 10 may be replaced by another having similar structure and / or chemical properties (e.g., hydrophobicity or hydrophilicity), that is, a conservative amino acid change, as long as the substitution is not substantially altered. protein function 5 or folding. Such substitutions are well known and understood in the art. Examples of these amino acid properties divided into different groups are hydrophobic (Met, Ala, Val, Leu, Ile), neutral hydrophilic (Cys, Ser, Thr), acidic (Asp, Glu), basic (Asn, Gin, His, Lys, Arg). , amino acids that affect chain orientation (Gly, Pro) and aromatic (Trp, Tyr, Phe) 10 amino acids. Substitutions within the groups in question are unlikely to result in significant changes in the structure of the polypeptide chain backbone (e.g., fold or helical conformation), charge, or hydrophobicity or side chain size of the molecule.

The BMP homologues of the present invention (e.g., BMP-4) comprise, for example, any bone morphogenetic protein containing or modified to contain at least one of said key amino acids, Pro6 or Pro21, or equivalent amino acids in a homologous BMP protein with numbering. is different. Any currently unknown BMP-4 protein of any of the 20 species modified as described above is also within the scope of this invention.

• ·

* * I

• · · • · • *. Because Pro21 replaces highly conserved leucine, in one embodiment of this invention, the BMP protein, or a homologue, derivative or fragment thereof, contains the amino acid proline corresponding to Pro21, or another. in an embodiment, the prolines corresponding to both prolines are Pro 6 and Pro 21, as defined at the respective positions of SEQ ID NO: 1. These sites • · * ··· 'are calculated from the amino terminal end of SEQ ID NO: 1, which corresponds to virtually any mature BMP-4 protein. The amino terminal end:.: The Y 30 sequence may be SPKHH (as in SEQ ID NO: 1) or a homologue thereof such as: ... · for example, a human, a reindeer or another mammal. These places can be; ! ·. determines by aligning the "Y" sequences of the homologues, derivatives or fragments in question with the sequence shown in SEQ ID NO: 1.

* ·

Ml ·· * '·· 35 If the amino acid sequence of the homologue, derivative or fragment in question has *: * ·: addition or deletion of any amino acid that affects numbering, this must be taken into account when defining the positions of said essential proline, e.g., by aligning sequences as above followed by 11937311 by determining the positions of the relevant amino acids. However, any of the homologues, derivatives or fragments of any of the BMP-4 proteins in question should substantially include the function and efficacy disclosed herein. Since all known BMP-4 proteins are well conserved (Table 1, at least 96% homology at amino acid level in mammals, or aligned), the positioning of these essential proline is unambiguous, as is the case with human BMP-4. These sites can also be readily determined from other BMPs (see alignment above). In general, the degree of homology at the amino acid level may be at least 70%, preferably 80%, more preferably 90%, and most preferably 96%.

In one embodiment of the invention, BMP is any BMP, or homologue, derivative or fragment thereof, containing the consensus sequence between two proline of the BMP-4 family: P-Q-R-A / S-R-K-K / R-N / K-K / R-N / H-C-15 R-R-H-S / A. In another embodiment, the BMP of the invention is any BMP or a homologue, derivative or fragment thereof which contains the corresponding consensus sequence of BMP-4 and BMP-2: PQRA / SR / KK / RK / N / R / L · N / MM / R / S N / H / SCR / KRHS / AP. This consensus sequence has been determined from the alignment alignments of several different species of BMP-4 and / or BMP-2 proteins, as shown in the ClustalX alignment below. Said consensus sequence corresponds to amino acids 6-21 of SEQ ID NO: 1. Other: Y: consensus sequences may also be defined, for example one which defines the region around another proline (Pro21 in SEQ ID NO: 0 1), · · · ·: as in one embodiment: CR / KRHS / APYVDFSD, or • · · 25 similar sequences that differ in length, for example, with 1-5 · · * Y amino acids. Corresponding consensus sequences can be determined from the following alignment or similar alignments made by aligning various related BMP proteins. Aligned BMP sequences are from reindeer, human, rabbit, cattle, dog, Texas deer (Cervus dama, DAMDA),:.: * .: 30 rats, mice, Asian shepherds (suncus murinus), chickens, • · · African claw frogs ( Xenopus laevis) and zebrafish (danio rerio).

In one embodiment, the BMP is any BMP-2, such as the reindeer BMP-2, • · *. * ', Modified to include those described in the text of the present invention. *** 35 amino acids or a sequence such as a consensus sequence.

• * 1 1 9373 12

BMP4_REINDEER SPKHHPQRARKKNKNCRRHSPYVDFSDVGWNDWIVAPPGYQAFYCHGDCPFPLADHLN BMP4_HUMAN SPKHHSQRARKKNKNCRRHSLYVDFSDVGWNDWIVAPPGYQAFYCHGDCPFPLADHLN

BMP4_RABBIT SLKHHPQRARKKNKNCRRHALYVDFSDVGWNDWIVAPPGYQAFYCHGDCPFPLADHFN

BMP4_B0VINE SPKHHPQRARKKNKNCRRHSLYVDFSDVGWNDWXVAPPGYQAFYCHGDCPFPLADHLN

5 BMP4_D0G SPKHHAQRARKKNKNCRRHSLYVDFSDVGWNDWIVAPPGYQAFYCHGDCPFPLADHLN

BMP4_DAMDA SPKHHPQRARKKNKNCRRHSLYVDFSDVGWNDWIVAPPGYQAFYCHGDCPFPLADHLN

BMP4_RAT SPKHHPQRSRKKNKNCRRHSLYVDFSDVGWNDWIVAPPGYQAFYCHGDCPFPLiADHLN

BMP4_M0USE SPKHHPQRSRKKNKNCRRHSLYVDFSDVGWNDWIVAPPGYQAFYCHGDCPFPLADHLN

BMP4_SUNCUS SPKHHPQRARKKNKNCRRHSLYVDFSDVGWNDWIVAPPGYQAFYCHGDCPFPLADHLN

10 BMP4_CHICKEN SPKHHG — SRKNKKNCRRHALYVDFSDVGWNDWIVAPPGYQAFYCHGDCPFPLADHLN

BMP 4 _X ENO PUS SPKQQR — PRKKNKHCRRHSLYVDFSDVGWNDWIVAPPGYQAFYCHGDCPFPLADHLN

BMP4_ZEBRAFXSH SPKQRG --- RKRNRNCRRHALYVDFSDVGWNDWIVAPPGYQAYYCHGECPFPLADHLN

BMP2_HUMAN QAKHKQ — RKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLN

BMP2_DAMDA QAKHKQ — RKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLN

15 BMP2 _MOUSE QAKHKQ - RKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLN

BMP2_XENOFUS QARHKQ - RKRLKSSCRRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLN

... -. . ★. ** 'ft ******************. *. ****. ******** - 1

BMP4_RE INDEER STNHAIVQTLVNSVNS SIPKACCVPTELSAISMLYLDEYDKWLKNYQEMWEGCGCR

20 BMF4_HUMAN STNHAIVQTLVNSVNSSI PKACCVPTELSAI SMLYLDEYDKWLKNYQEMWEGCGCR

BMP4_RABBIT STNHAIVQTLVNSVNSSI PKACCVPTELSAI SMLYLDEYDKWLKNYQEMWEGCGCR

: V: BMP4_BOVINE STNHAIVQTLVNSVNSSI PKACCVPTELSAI SMLYLDEYDKWLKNYQEMWEGCGCR

• ·

• · 1; BMP4_DOG STNHAIVQTLVNSVNSSI PKACCVPTELSAI SMLYLDEYDKWLKNYQEMWEGCGCR

• · · ·

.1. I BMP4_DAMDA STNHAIVQTLVNSVNSSI PKACCVPTELSAI SMLYLDEYDKWLKNYQEMWEGCGCR

• · ·

: 25 BMP4_RAT STNHAIVQTLVNSVNS SI PKACCVPTELSAI SMLYLDEYDKWLKNYQEMWEGCGCR

• · 1

**. ' BMP4_MOUSE STNHAIVQTLVNSVNSSI PKACCVPTELSAI SMLYLDEYDKWLKNYQEMWEGCGCR

* · · • · ·

BMP4_SUNCUS STNHAIVQTLVNSVNSSI PKACCVPTELSAI SMLYLDEYDKWLKNYQEMWEGCGCR

• ·

· ** BMP4_CHICKEN STNHAIVQTLVNSVNSSI PKACCVPTELSAI SMLYLDEYDKWLKNYQEMWEGCGCR

BMP4 XENOPUS STNHAIVQTLVNSVNSSI PKACCVPTELSAI SMLYLDEYDKWLKNYQEMWEGCGCR

• · · _ _

30 BMP 4_Z EBRAFISH STNHAIVQTLVNSVNTNIFKACCVPTELSAISMLYLDETDRWLKNYQEMWEGCGCR

* · 1

*, t, · BMP2_HUMAN STNHAIVQTLVNSVNSKI PKACCVPTELSAI SMLYLDENEKWLKNYQDMWEGCGCR

I. . BMP2_DAMDA STNHAIVQTLVNSVNSKI PKACCVPTELSAI SMLYLDENEKWLKNYQDMWEGCGCR

• · · • · 1 ·

β 1 1 1 φ BMP2_MOUSE STNHAIVQTLVNSVNSKI PKACCVPTELSAI SMLYLDENEKWLKNYQDMWEGCGCR

• ·

BMP2_XENOPUS STNHAIVQTLVNSVNTNIPKACCVPTELSAISMLYLDENEKWLKNYQDMWEGCGCR

• · * 1 «- j · · ** ★★ *** ★ 1 ★ ** · 1 · **. ********************* ·· ******* «*********« 119373 13

In one embodiment, the BMP is any BMP, or homologue, derivative or fragment thereof, comprising amino acids 6-21 of SEQ ID NO: 1. In another embodiment, the BMP of the present invention is any BMP, or homologue, derivative or fragment thereof, comprising amino acids 5-16 of SEQ ID NO: 1. In yet another embodiment, the BMP of the present invention is any BMP or homologue, derivative or fragment thereof comprising amino acids 16-23 of SEQ ID NO: 1. In another embodiment, the BMP of the present invention is any BMP, or homologue, derivative or fragment thereof, comprising amino acids 1-23 of SEQ ID NO: 1. In another embodiment, the BMP of the present invention is a BMP-4 protein, or a homologue, derivative or fragment thereof, such as that comprising the amino acid sequence of SEQ ID NO: 1. The homologues, derivatives, or fragments mentioned in these embodiments must contain at least amino acid Pro6 (as exemplified by the mutation L-P as shown in the above alignment) or both Pro6 and Pro21 as described above. Said homologues, derivatives, or fragments do not include known BMP-4 proteins as such, such as human BMP-4, since they do not contain those amino acids characteristic of the BMP-4 protein of the present invention. However, if the known BMP-4 is modified to contain at least one of these characteristic amino acids, it can be considered as such a homologue, derivative or fragment.

An embodiment of the present invention provides a nucleic acid molecule such as:. A DNA or RNA molecule encoding said BMP of the present invention.

Due to the degeneracy of the genetic code, there are several different nucleic acid sequences encoding the BMP of the invention. All such: * V nucleic acid variants are within the scope of this invention. Preferably said nucleic acid molecule is a DNA molecule. Examples of said DNA sequences are shown in Figures 5-7.

:.: V 30 An embodiment of the present invention provides a replicable vector comprising: ** [: the nucleic acid molecule described above, operably linked to:] ·. a sequence that controls its expression. Such vectors may be used to produce recombinant BMPs of the invention in a suitable **. ** 'host system.

35:: **: The nucleic acid encoding the BMP of the present invention can be inserted into that replicable vector for cloning or expression. Several vectors are publicly available. The vector may be, for example, in the form of a plasmid, cosmid, viral particle 119373 14, or phage. The appropriate nucleic acid sequence may be inserted into a vector using a variety of procedures. Generally, DNA is ligated to a suitable restriction endonuclease cleavage site using methods well known in the art. The vector may be, for example, one or more signal sequences, origin of replication, one or more marker genes, enhancer element, promoter and transcription termination sequence. Construction of such suitable vectors containing one or more of these components utilizes standard ligation techniques well known to those skilled in the art.

10

In general, the BMP in question can be produced by gene expression by expression in any suitable host cell, such as a bacterial cell. Such methods are well known in the art and can be found in the literature. It is essential that the protein folds into the correct form during expression and that it contains all necessary post-translational modifications.

It is not always possible to produce and purify certain proteins properly, for example due to problems with solubility and refolding. In general, E. coli cannot make the post-translational modifications that are typical of mammalian cell systems. However, the present inventors have produced the mature portion of reindeer BMP-4 as a recombinant protein in E. coli and, after purification and refolding, have shown it to be in a biologically active form.

An embodiment of the present invention provides a host cell comprising a? T * 5 V nucleotide molecule or nucleotide vector described above. Useful :: i .: Cells are all prokaryotic and eukaryotic cells capable of expressing the protein of this invention. Such host cells are well known in the art and one of ordinary skill in the art can readily select a suitable host cell. One embodiment provides BMP produced by growing said cells to express the protein in question and recovering said produced: J *. protein from the host cell in question. Any useful method] ··· [can be used for protein recovery and purification and such · · * ”methods are well known in the art.

The BMP of the present invention may be used in the treatment of disorders affecting bone, cartilage, tendon or tooth defects or diseases, or the like where regeneration, repair, or growth is desirable, or other 119373 15 diseases. The protein of the present invention may also be used for wound healing such as burns, surgical wounds and stomach ulcers and similar tissue repair as well as for the treatment of cancer as disclosed in EP1131087. Because BMP generally lacks species specificity, the patient suffering from the defect in question may be any suitable animal, preferably a mammal such as a human, and the BMP protein used in the treatment may be derived from any suitable source. The use of similar BMP proteins in many therapeutic applications is well known in the art (see, for example, US 6245889 and WO 98/51354).

10 '' Disorders Related to Bone, Cartilage, Tendon or Tooth Defects' as used herein generally refers to any disorder in which the healing or rebuilding, i.e. regeneration, of bone, cartilage, tendon or teeth is desirable. Non-limiting examples of treatment measures for bone, cartilage, tendon or dental disorders or diseases, or the like, are bone and dental regeneration, repair, and growth; bone regeneration, repair and growth in mammals such as man; treatment measures to correct abnormalities in bone formation or regeneration; wound healing, ectopic bone induction, and healing of segmental bone damage in vertebrates; treatment measures to correct skeletal disorders and deformities; repair of major bone damage due to trauma, neoplasm or congenital:. malformations, reduced due to implanted internal prosthesis • · ·! reconstruction of bone stores in repair operations or retardation of bone fractures • * ·. * / 25 or unrelated bone fractures; repair of bone and cartilage defects i * V, such as' 'critical size defectif', '' non-critical size defects' ', • unrelated fractures, segmental unrelated fractures; acute fractures, cartilage defects, bone-cartilage defects, cartilage bone defects, local bone and cartilage formation; defects due to degenerative diseases; dental applications for repairing dental tissue, alveolar bone, dental cement, tooth root membrane, filling tooth root canal contents, developing and improving dental implant attachment. More examples of these disorders can be found in Ann Rheum Dis, Volume 62, 2003, **: '73-78: Reddy AH: Cartilage Morphogenic Proteins: Role in Joint Development, 35 Homeostasis and Regeneration.

• ·

Other diseases where the BMP of the present invention is useful include cancer, fibromyalgia, psoriasis and other dermatological disorders, and rheumatic disorders 11937316 and the like. Examples of such cancers and methods and compositions for treating them are disclosed in EP1131087.

One embodiment of the invention provides a BMP, such as BMP-4, for use in any of the embodiments described herein with one or more other morphogenetic proteins, such as another type of BMP or the like. Generally, this achieves synergy benefits, as is known in the art. Examples of other suitable BMP proteins include, but are not limited to, BMP-1, BMP-2, BMP-3, other BMP-4, BMP-5, BMP-6, BMP-7 and BMP-8. Other therapeutically useful agents may also be used, such as epidermal growth factor, fibroblastic growth factor and transforming growth factor (US 6,245,889). In one embodiment of the invention, the complementary morphogenetic protein in question is derived from a reindeer, like any other reindeer BMP protein. In one embodiment of the invention, BMP-4 is provided for use as a dimer, homodimer, or heterodimer in combination with another BMP protein as described above. In another embodiment, BMP-4 is provided for use in a dimeric form or in combination with another agent or protein as described above for the manufacture of a medicament for the treatment of disorders as described in the specification.

20

One embodiment of the invention provides an osteogenic device, such as an implant, containing the BMP of the present invention. The osteogenic device may include a biocompatible matrix, such as calcium phosphate, • · T: carboxymethylcellulose, collagen matrix, or combinations thereof. In one embodiment, the calcium phosphate matrix is a hydroxyapatite matrix.

This matrix may allow the slow release of BMP and / or: an appropriate environment for the release of BMP. The osteogenic device may also include a metallic implant surrounded by said biocompatible matrix. One example of this metal is titanium. Some examples of such osteogenic devices are disclosed in WO 98/51354.

* * · • · • * • · ·. Non-limiting examples of various supports, carriers, or supports for forming, for example, various osteogenic devices or the like, containing the protein of this invention, are expressed in the following various forms: powder, sponge, tab, membrane, gel, web or solution or ·: ··· suspension; semi-solid solution carrier for use in intramuscular, intravenous, bone marrow, or intra-articular injection; isolated mesenchymal stem cells; any pharmaceutically acceptable vehicle; acceptable as a matrix, a material selected from the group consisting of hydroxyapatite, collagen, polymers (e.g., polymers of polylactic acid and polyglycolic acid), synthetic polymers, hyaluronic acid, α-BSM, calcium phosphate, tricalcium phosphate, biopolymer, non-porous resorbable biopolymer, coral, demineralized bone, bio glass, any biodegradable material and combinations thereof; binding agents selected from the group consisting of mannitol, dextrans, white petrolatum, alkyl and methyl celluloses, wetting agents such as sodium salts, fobrin glue, fibrinogen and thrombin of mammalian origin, and combinations and mixtures thereof. The osteogenic device may be, for example, a structurally stable, three-dimensional implant in the shape of a cube, cylinder or block or anatomically shaped or injectable. Examples of osteogenic devices, useful materials and techniques are disclosed in Skeletal reconstruction and 15 bioimplantation (T. Sam Lindholm, 1997, Springer-Verlag, Heidelberg, Germany).

One embodiment of the invention provides a pharmaceutical composition comprising a therapeutically effective amount of BMP of this invention in a pharmaceutically acceptable carrier or carrier. Such pharmaceutical compositions 20 may be used to treat disorders associated with bone, cartilage, tendon or tooth damage or other diseases, wounds and other tissue damage, or any of the disorders described herein.

An embodiment of the invention provides a method for inducing the formation of bone, cartilage, tendon, tooth, or the like, wherein said bone, j * y cartilage, tendon, tooth, or the like is treated with the BMP of this invention or combinations thereof as described above, in vitro or in vivo. Another embodiment of the invention provides a method of treating the disorders described in the specification, comprising administering an isolated BMP of the invention to a patient suffering from such disorders. The BMP in question may be administered in the form of a pharmaceutical: [[[:: composition or osteogenic device as described above.

:) ·, Complementary morphogenetic proteins or other useful substances may be administered in conjunction with the BMP of the present invention, as described '*; ** above, to enhance the therapeutic effect.

The following description and examples describe how the mature portion of recombinant reindeer-BMP-4 according to one embodiment of the invention was produced in E coli. After purification and refolding, osteoinductive 119373 18 activity was confirmed by bioassay in the mouse hindlimb muscle pocket. This in vivo bioassay has been a standard method for determining BMP activity since the discovery of the protein. It involves BMP implantation of mouse hind foot muscle and assessment of heterotropic new bone induction radiologically 5 and histologically 10 to 21 days after implantation.

In the assay of BMP activity, image analysis is performed by transmitting an X-ray through the bone, which is seen on the film as an X-positive darkening when compared to soft tissue. This allows for radiographic detection and radiomorphometric quantitation of 10 newly formed bone obtained by implanting BMP or other bone inducing agent at a heterotopic or orthotopic site.

Osteoinductivity was observed in all three groups studied (1 mg, 3 mg, 15 and 5 mg recombinant reindeer BMP-4) and the response increased with dose (Table 2). When compared to human recombinant BMP-4 protein, reindeer BMP-4 was approximately five times more potent inducer of bone formation than similarly produced recombinant human BMP-4 protein (up to 7.5 mg dose) and is therefore potent osteoinductive. substance for clinical applications.

• φ • · • »·: 1: 1 ma 3 ma 5mq • · · - • M · • · • * ·: *, · * Poro (+) ++ + (+) (+) +++ ++ + ++ +++ +++ _i ± L_i ±) __: __: __ ± __ ± __ω __: __ = __: __ ω__i_ · * :: / __: __: __: __: __: __-__: __ ++ (+) + +++ • · ··· * __ ^ __-__ ++ - +++ +++ +++ +++ +++ _____ (+) __ "__ +++ + ++ +++ +++ +++ __ ~ • · * • · · ···. _ - - -.....

• · · • ·

···. I I | I I ----- I

•, ·. Human (+) - (+) - - + • · · • · · · · · · · · - ...... ..II .. I ......... * .... ....... · * ····································································································· os 25 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. -4 protein. 119373 19 was performed as described in Marshall R. Urist, J.J. Chang, A. Lietze, Y.K. Huo, A.G. Brownell and J. DeLange (1987): Preparation and Bioassay of Bone Morphogenetic Protein and Polypeptide Fragments, Methods Enzymol 146: 294-312.

5

eXAMPLES

Example 1: Cloning and Sequencing of the mature portion of reindeer BMP-4 cDNA 10 A. RNA Isolation

Three-year-old male reindeer horns were severed and frozen in liquid nitrogen immediately after slaughter. The frozen horns were cut into 0.5 cm pieces and stored at -70 ° C. Reindeer horn mRNA was isolated using the QuickPrep® Micro mRNA Purification Kit (Pharmacia Biotech). Part of the reindeer horn piece was cut into small pieces (about 1 mm 3) and 0.1 g of this tissue was added to 0.6 ml of Extraction buffer containing guanidine thiocyanate and N-lauroyl sarcosine. The tissue was homogenized on ice with Ultra Turras three times for 20 to 10 seconds and cooled between homogenization runs. 1.2 ml of Elution buffer was added and the suspension was homogenized once more for 10 seconds. The result was a uniform suspension.

• · • · · * T | The reindeer horn homogenate and the Oligo (dT) cellulose were centrifuged at full speed / 25 [14,000 rpm, room temperature, Centrifuge 5415 C (Eppendorf)] for 1 minute. The buffer on top of the Oligo (dT) cellulose pellet was removed and the ··· clear tissue homogenate was pipetted in its place. The tube was turned upside down so that I ··

The oligo (dT) cellulose pellet would be made into a suspension. The suspension was gently stirred for five minutes and centrifuged at full speed [14,000 rpm, 30 at room temperature, Centrifuge 5415 C (Eppendorf)] for ten seconds. The supernatant was discarded.

IM

• ♦ * · ·

Oligo (dT) cellulose was resuspended in High-Salt Buffer [10 mM

Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 M NaCl] and the suspension was centrifuged at full speed 35 [14,000 rpm, room temperature, Centrifuge 5415 C (Eppendorf)] ·: ·· · For ten seconds. Washes with High-Salt Buffer buffer were repeated five times and additionally twice with Low Salt Buffer Buffer [10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 M NaCl]. Three ml of Low Salt Buffer buffer was added and the suspension was loaded on a MicroSpin column. The MicroSpin column was placed in an Eppendorf tube and centrifuged at full speed for five seconds. Column Oligo (dT) cellulose was rinsed three times with Low Salt Buffer.

5 Reindeer Horn mRNA was eluted from the MicroSpin column into a clean Eppendorf tube by adding 0.2 ml of 65 ° C Elution Buffer (QuickPrep® Micro mRNA Purification Kit, Pharmacia Biotech) and centrifugation at full speed [14,000 rpm, room temperature, Centrifuge 5415 Eppendorf)] for five seconds. The elution step was repeated twice. The isolated mRNA was precipitated by adding 10 µl of each glycogen solution (5-10 mg / ml in DEPC-treated H 2 O), 1/10 volume of K-acetate solution (2.5 M potassium acetate, pH 5.0) and 0.5 ml of absolute ethanol (cooled to -20 ° C). Precipitation was allowed to proceed at -20 ° C for at least 30 minutes and the mRNA was centrifuged at full speed [14,000 rpm, 4 ° C, Centrifuge 5415 C (Eppendorf)] for 5 minutes. The precipitated mRNA was stored at -70 ° C until cDNA synthesis was performed.

B. cDNA Synthesis Reverse transcription of reindeer horn mRNA was performed by modification of the Time Saver ™ cDNA Synthesis Kit (Pharmacia Biotech). 3 µg of mRNA were heat denatured at 65 ° C for ten minutes and chilled on ice. 0.2 pmol of DTT, 0.5 µg of Oligo (dT) 12-I primer and heat denatured mRNA were added to a First Strand reaction mixture containing: FPLCpure ™ Cloned // Murine Reverse Transcriptase, RNAguard ™, RNase / DNase-Free BSA, dNTPs

i *: s: (dATP, dCTP, dGTP, and dTTP) in aqueous buffer (Time Saver ™ cDNA

(Synthesis Kit, Pharmacia Biotech). The mixed solution was incubated at 37 ° C for one hour. for an hour. After incubation, the First Strand reaction mixture was added to the Second Strand reaction mixture containing E. coli RNase H and E. coli DNA polymerase 1 and 30 dNTPs in aqueous buffer (Time Saver ™ cDNA Synthesis Kit, Pharmacia Biotech). The solution was gently mixed and incubated at room temperature for 30 minutes. \ minutes. The synthesized cDNA was stored at + 4 ° C.

C. Mapping of Reindeer Horn cDNA 9: · .. 35 e ·: ··· The mature portion of reindeer BMP-4 cDNA was amplified by PCR (Polymerase chain). reaction) using primers (5'-GGATCCGAGCCCCAAGCATCACCCACAGAGG -3 ') 21 119373 and (3'-AAGCTTGCGGCACCCACATCCCTCCACTAC - 5') (Table 3) designed based on the homology of the already known Texas six-deer BMP-4 gene. The PCR reaction mixture (50 µl) contained 100 ng of reindeer horn cDNA, 40 pmol of each primer, 0.4 mM dNTP (PCR Core Kit, Roche) and 0.7 5 U / μΙ Expand High Fidelity Enzyme (heat-stable Taq). Polymerase + Proofreading Polymerase, Roche) in Expand High Fidelity Buffer containing MgCl 2 (Expand High Fidelity PCR System, Roche). The reaction was performed using a Mastercycler personal apparatus (Eppendorf) according to the following program: initial denaturation at 94 ° C for 4 minutes, and the following 25 cycles: 10 denaturation at 94 ° C for 1 minute, primer attachment at 55 ° C for 1 minute, extension of DNA chains C 2 minutes. The final extension was performed at 72 ° C for 10 minutes.

Primers used in cloning_

Mature portion of reindeer BMP-4 cDNA_ pTrcHis2A 51-> 3 'GGATCCGAGCCCCAAGCATCACCCACAGAGG____'5' AAGCTTGCGGCACCCACATCCCTCCACTAC_ PIVEX2.4 5 '- + 3' CCGCGGTAGCCCAG

3'— »5 'GGATCCTAGCGGCACCCACATCCCTCCACTAC

15 • · v Table 3. Primers used for cloning the mature part of reindeer BMP-4 D. Cloning into pGEM-T® vector mm 1 · · · · ·: 20 PCR the product was purified using the Wizard® PCR Preps DNA Purification System “1 ·! (Promega) and ligated into the pGEM-T® vector (Figure 1) using T4 DNA ligase (pGEM-T® Vector System I; Promega). 0.3 µg of purified PCR product and 2.3 µg / ml pGEM-T® vector were added to the ligation buffer containing 18 mM Tris-HCl (pH 7.8), 6 mM MgCl 2, 6 mM DTT, 0.3 mM ATP, 3% polyethylene glycol and * ··· 1 25 0.14 U / μΙ T4 DNA ligase at a total volume of 66 μΙ. The reaction was allowed to: take place in a water bath of + 16 ° C, which cooled to + 4 ° C overnight. Born, New ··· ·: 1 ··. the plasmid was called: pGEMrd4 / 116 (Figure 1).

E. E. Preparation of Competent Escherichia coli TOP 10 F 'Cells

Competent Escherichia coli TOP 10 F cells were prepared by the calcium chloride / magnesium chloride method. 2 ml of LB broth were inoculated with 5 E. Coli TOP 10 F cells and grown overnight at 37 ° C with shaking (225 rpm). The next morning, 100 ml of fresh LB broth was added to 1 ml of overnight culture and grown with shaking (225 rpm) at 37 ° C until OD 600 = 0.5-0.6. Cultured cells were harvested by centrifugation (2.500 x g, 5 minutes), resuspended in 10 ml of 0.1 M MgCl 2 solution and re-collected by 10 centrifugation (2.500 x g, 5 minutes). After MgCl2 treatment, cells were suspended in 10 in 0.1 M CaCl2 solution, incubated in an ice bath for 30 minutes, and collected again by centrifugation (2.500 x g, 5 minutes). The CaCl 2 treatment was repeated, however, with a second run of 3.5 mL of CaCl 2 and incubation of the cells for one hour. Glycerol (final concentration 14% (v / v)) was added to the suspension and the suspension was divided into 200 μΙ aliquots. Competent E. Coli TOP 10 F 'cells were frozen in liquid nitrogen and stored at -70 ° C.

F. Transformation of competent Escherichia coli TOP 10 F 'cells and selection of clones containing reindeer BMP-4.

20

Competent Escherichia coli TOP 10 F cells were thawed in an ice bath for 15 minutes

: Y: time. 10 μΙ of ligation mixture (described above) was added to 100 ph of TCM solution (10 mM

• ·; Tris-HCl, 10 mM CaCl2, 10 mM MgCl2, pH 7.0) and mixed with 200 μΙ. : competent E. coli cells. The mixture was incubated on ice for 30 minutes before • · ·

25 heat shocks (43 ° C, 3 minutes). After heat treatment, 800 μΙ LB was added

The culture broth and cells were allowed to recover at 37 ° C for 45 minutes. Transformed;: i .: Cells were harvested by centrifugation at full speed for 2 minutes and resuspended in · · · · ··· 30 phage broth. The cell suspension was plated on two LB plates containing 25 pg / ml ampicillin, 1 mmol IPTG (isopropyl-pD-thiogalactopyranoside) and 2.4 nmoles of X-gal (5-bromo-4-chloro-3-indolyl-pD-): ... · 1 galactoside) and cells were grown in plates overnight at 37 ° C. Positive clones:! ·. were found to be white in color due to a · complementation of the IacZ gene • · [··· [. The method is described in detail in Sambrook and Russel (2001) Molecular Cloning, Cold Spring Harbor Laboratory Press, New York.

· * · • ·· 119373 23 Isolation of G. pGEMrd4 / 116 Plasmids and Sequencing of cDNA Inserts

Plasmids were isolated using the Wizard® Plus Minipreps DNA Purification System kit (Promega) and further purified by ethanol precipitation. The identity of the cDNA was confirmed by sequencing on an ABI Prism (Perkin-Elmer

Corporation). The sequencing reaction was performed using a DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech) and a Mastercycler Personel PCR Device (Eppendorf). The PCR primers used for sequencing were (5'-T AATACGACTCACT AT AGGGCGA-3 ') and (3'-10 ATTTAGGTAACACTATAGAATAC-5') (Table 4) and program used: 25 cycles of denaturation at 94 ° C for 30 seconds, primer addition at 50 ° C 15 seconds, chain extension 60 ° C. Amplified PCR products were mixed with ethanol. To 10 µl of the reaction mixture was added 1 µl of 1.5 M Na acetate - 250 mM EDTA buffer and 95-100% ethanol to a final concentration of 75% ethanol. Precipitation was allowed to occur in an ice bath for 10 minutes and then the mixture was centrifuged for 20 minutes. The supernatant was discarded and the precipitate was washed at room temperature with 125 silicon 70% ethanol. The mixture was centrifuged rapidly and the ethanol used for washing was removed as efficiently as possible. The precipitate was dried at 37 ° C for a few minutes until all ethanol had evaporated. 20 The ABI Prism equipment was in the Department of Medical Biochemistry and Molecular Biology at the University of Oulu and the actual sequencing was performed there.

0 9 0 0 9 0 0 9 - · ·: Primers used for sequencing_

pGEM-T® plasmids 5 '-> 3' TAATACGACTCACTATAGGGCGA

3 '^ 5' ATTTAGGTGACACTATAGAATAC

• · · 0 09 9 099

* ···: pTrcHis2A 5 '-> 3' AGAGGTATATATTAATGTATCG

plasmids 3 '^ 5' ATGGTCGACGGCGCTATTCAG

* * · · · · · · ·

0 5'— ► 3 'TAATACGACTCACTATAGGGCGA

:. * ·. plVEX2.4 plasmids 3 '^ 5' GCTAGTTATTGCTCAGCGG

1 · «· • · · • • • * ·· A {.

25 • 1 Table 4. Primers used for sequencing the mature part of reindeer BMP-4 9 9 9 119373 24

Example 2. Expression of the recombinant mature protein of reindeer BMP-4 in Escherichia coli TOP 10 F ', Origami B (DE3) and Rosetta (DE3) cells A. Subcloning of the mature portion of reindeer BMP-4 from pGEM-T® 5 expression vector pTrcHis 2A (Invitrogen) and transformation into competent Escherichia coli TOP 10 F cells.

Subcloning of the mature portion of reindeer BMP-4 from the pGEM®-T vector expressing vector pTrcHis 2A (Figure 2) was accomplished by removing the mature portion of the 10 pGEMrd4 / 116 vector with Bam HI and Hind III restriction enzymes and ligating the insert into pTrcHis 2A. with the same enzymes. Digestion of pGEM®-T and pTrcHis 2A (1 µg) with Bam HI (Roche) and Hind III (Roche) was performed in 10 µl of 10 mM Tris-HCl, 10 mM NaCl, 5 mM MgCl 2, 1 mM mercaptoethanol, pH 8.0 (SuRE / Cut B, Roche) using 1 U / μΙ each of 15 restriction enzymes. The reaction was allowed to proceed for 1.5 hours at 37 ° C, after which the restriction enzymes were inactivated at 65 ° C for 20 minutes and frozen at -20 ° C. The ligation (0.1 U / μg ligase concentration) was performed in 2X Rapid Ligation Buffer (supplied with pGEM-T® vector (Promega)) in a +16 ° C water bath which was allowed to cool slowly to + 4 ° C overnight.

20

The new construct was verified by sequencing (the method is described in Example 1, section G) using primers: (5'-AGAGGTATATATTAATGTATCG -3 ') and: (3'-ATGGTCGACGGCGCTATTCAG-5'). An expression vector containing the cDNA of the mature portion of pTrcHis! \: 2A and reindeer BMP-4 was called: pTrcrd4 / 116 (Figure • ··. *, ·] 25 2). Competent Escherichia Coli TOP 10 F 'cells were transformed as described in Example 1, Part F.

• · ·

• «I

B. Insertion of the mature portion of the reindeer BMP-4 into the expression vector pET22b (+) (Novagen) and transformation into the competent Escherichia Coli Origami V: 30B (DE3) and Rosetta (DE3) - to cells • · · «· • · t ··:! ·. Subcloning of the mature portion of reindeer BMP-4 into an expression vector • * [· * · [pET22b (+) (Novagen) (Figure 3) was performed as described above (see Example 2, Part A). The resulting new plasmid containing the pET22b (+) vector and ··: * ·· 35 mature part of the cDNA molecule of reindeer BMP-4 was called '' * i under the name: pETrd4 / 116 (Figure 3). Competent Origami B (DE3) and Rosetta (DE3) cells were transformed according to the manufacturer's instructions (Novagen).

119373 25 C. Expression of the mature portion of the reindeer BMP-4 recombinant protein in Escherichia coli cultures and cell harvesting in E. coli cells [TOP 10, Origami B (DE3) or Rosetta (DE3)] containing either 5 pTrcrd4 / 116 or pETrd4 / 116 plasmid was grown overnight in 50 ml SOB medium containing ampicillin (50 pg / ml) and Rosetta (DE3) cell culture also chloramphenicol (35 pg / ml) at 37 ° C with shaking (225 rpm). . The next morning, 24 ml of overnight culture was added to 1200 ml of SOB medium containing the above antibiotics and culturing was continued at 37 ° C with shaking (225 rpm) until Οϋβοο was 0.6, with cells in the mid-log phase of growth. round. At this point, the induction of the recombinant protein was accomplished by the addition of IPTG to a final concentration of 1 mM. After induction, cells were grown for four to five hours, after which they were harvested by centrifugation. The amino acid sequences of the recombinant proteins derived from the nucleotide sequences are shown in Figure 5 (pTrcrd4 / 116) and Figure 6 (pETrd4 / 116).

Example 3: Purification and folding of the mature portion of reindeer BMP-4 recombinant protein 20 A. Washing of inclusion bodies The harvested cells were suspended by shaking in 50 mM Na phosphate buffer, pH 7.0, 220 g [ · [| Cells / liter of buffer). The suspension was centrifuged at 5500 x g for 45 minutes at + 4 ° C.

The washing with Na phosphate solution was repeated once. The cell pellet was weighed and stored at -70 ° C overnight. A frozen pellet with partially broken cells was thawed and · ·

** was suspended (25 mg / ml) in 20 mM Tris-HCl buffer, pH 8.5 containing 0.5 mM

* ···: EDTA, shaking for 2 minutes. The suspension was centrifuged at 26,000 x g for 30 minutes at + 4 ° C and the Tris-HCl-EDTA wash was repeated once. The resulting cellular precipitate 30 was weighed. After the final wash, the cell pellet was suspended (35 mg / ml) with shaking

(200 rpm overnight, at room temperature) in lysis buffer (6 M GuHCl-20 mM)

:, * ·. Na-phosphate-0.5 M NaCl, pH 8.0), with the remainder of the whole £. coli cells • * · *. 'V are fragile and the inclusion bodies are soluble. The suspension was centrifuged at * "** (26,000 xg, 45 minutes at room temperature), the precipitate discarded, leaving ··: * .. 35 recombinant protein soluble in the supernatant. Finally, the supernatant ·: ··: filtered through a 45 µm filter to ensure removal of all cell debris.

119373 26 B. Precipitation at an isoelectric point (pl)

The reindeer BMP-4 recombinant protein expressed from pETrd4 / 116 in Escherichia coli Origami B (DE3) or Rosetta (DE3) cells was mixed at 5 isoelectric points at pH 8.12. The isoelectric point was determined by computer calculation from the amino acid sequence of recombinant reindeer BMP-4 (Figure 6). The precipitate was collected by centrifugation (12,000 x g, 30 min, at room temperature) and suspended in lysis buffer (6 M GuHCl - 20 mM Na phosphate - 0.5 M NaCl, pH 8.0).

10 C. immobilized metal affinity chromatography (IMAC)

Escherichia coli cells were lysed by shaking with 6 M GuHCl - 20 mM Na-phosphate -0.5 M NaCl; (pH 8.0) in solution for two hours and filtered through a 45 µm filter. The IMAC method utilized pre-packaged HiTrap

Chelating HP affinity columns (Amersham Pharmacia Biotech). The columns were charged with Co2 +, Cu2 + or Ni2 + ions according to the manufacturer's instructions. The His-Tag epitope at the end of the rdBMP-4 protein was utilized such that the His-tag epitope bound to a column charged with metal ions and E. coli-derived cell debris flowed through the column. The filtered, post-wash supernatant was applied to the column surface after charging the column matrix. Most impurities: V: removed by washing the column with lysis buffer (6 M GuHCl - 20 mM Na phosphate - 0.5 M NaCl, pH 8.0) with 5-10 fold column volume. Second wash, * * · ·

5-10 fold column volume was performed with lysis buffer at 6M

:. ·] 25 GuHCI was replaced by 6 M urea. Recombinant reindeer BMP-4 was eluted from the HiTrap column using a pH gradient from pH 7.0 to pH 4.0 (6 M urea - 20 mM Na phosphate - 0.5 M NaCl). Fractions were analyzed by SDS -PAGE and the purest rdBMP-4 containing ψ · ***** fractions were pooled for folding the recombinant protein (Figure 8).

30 D. Folding of the mature portion of recombinant rdBMP-4 ··· • · · ···:! ·. BMP-4 fractions analyzed by SDS-PAGE were pooled and dialyzed with water * ···! against. Protein precipitated by dialysis was collected by centrifugation and suspended by incubation for 2 hours at 25 ° C in 8 M urea - 0.1 M Tris / HCl, pH 8, containing 1 * · 35 plus 100 mM DTT, 1 mM EDTA. The pH was lowered to pH 3-4 by the dropwise addition of 1 M HCl. The DTT was completely removed by dialysis for two hours at 25 ° C against 6 M urea-10 mM HCl. Dialysis was continued at + 4 ° C overnight against 6 M urea. Folding of recombinant rdBMP-4 was performed on 119373 27 biphasic dialysis. The first dialysis solution was 20 mM Tris-HCl - 150 mM NaCl - 3 M urea (pH 7.5). The dialysis buffer was changed four times over two or three days. In the second step, all salts were removed by thorough water dialysis. Dialysis water was changed at least six times over two, three days. The desalted sample was centrifuged and the pellet dried by lyophilization. At this point, BMP-4 was 75% pure and 50% folded as determined by non-reducing SDS-PAGE. The amount of folded dimer of reindeer recombinant BMP-4 was measured by densitometry on Coomassie Brilliant Blue stained gels.

10

Example 4. Testing the biological activity of the mature portion of reindeer recombinant BMP-4

The biological activity of the lyophilized reindeer BMP-4 recombinant protein was tested by implanting less than 1 mg of recombinant protein absorbed into a Lyostrypt® collagen blanket or gelatin capsule into the mouse thigh muscle pocket. BSA served as control. The hind legs were x-rayed and the implant sites were dissected and fixed in 10% neutral formalin solution. Fixed implants were cut into 4 µm sections and stained with hematoxylin-20 eosin solution. The sections were examined under a light microscope. New bone formation was evaluated by X-ray imaging by determining surface area and optical density. X-rays were transferred to a computer using an optical scanner (HP Scan Jet, Hewett-Packard, USA). The formation of ectopic and orthotopic new bone T i was evaluated by the area of calcified tissue seen in X-rays (mm 2) / 25 using Scion Image Beta 4.02 (Scion Corp., USA). The average optical density (mmAl) of a given region was measured by the same device. Optical Density: Calibration was performed using an aluminum plate (Al) with 0.25 · · · mmAl steps and a calibrated density giving a range up to 4 mmAl.

: 30 Example 5: Expression of the mature portion of reindeer BMP-4 recombinant protein

Rapid Translation System on the RTS 500 * ♦ · · · · * A. Construction of the RTS 500 Expression Vector pIVEX2.4c (Roche) 35 Amplification of the normal mature portion of reindeer BMP-4, PCR product · : ··· purification, ligation into pGEM-T® vector (Figure 1), transformation into competent Escherichia coli TOP10 F cells (Invitrogen), plasmid purification and insert sequencing were performed as described in Example 119373 28 except for reindeer BMP- The following primers were used to amplify the 4 mature sections: (5'-CCGCGGTAGCCCCAAGCATCACCCACAGAGG-3 ') and (3'-GGATCCTAGCGGCACCCACATCCCTCCACTAC-5') (Table 2) and the construct was called pGEMrd4 / 116/2 (pMEMrd4 / 116/2). The primers used to construct the construct were: (5'-TAATACGACTCACTATAGGGCGA-3 ') and (3'-GCTAGTTATTGCTCAGCGG-5') (Table 4). The primers used for amplification had a Ksp I (Sac II) restriction enzyme cleavage site at the 5'-end and a Bam HI restriction site at the 3'-end of the primer, and these sites were utilized for subcloning the mature portion of reindeer BMP-4. Plasmids pGEMrd4 / 116/2 and pIVEX 2.4c (0.5 µg) were digested using 10 mM Tris * HCl, 10 mM MgCl2, 1 mM dithioerythritol, pH 7 (SuRE Cut Buffer L, Roche) buffer and 1 U / μΙ, for a total volume of 10 µl. Restriction enzymes were inactivated before ligation and the ligation reaction was performed as described in Example 2, Part A. The new construct is termed plVEXrd4 (pMU500) (Figure 4).

B. Production of the mature portion of recombinant BMP-4 in RTS 500 The RTS 500 reaction was performed following the instructions of the Rapid Translation System RTS 500 E. coli Template Kit. The amino acid sequence and the corresponding nucleotide sequence of the recombinant protein are shown in Figure 7.

Results • · · •! Cloning of Reindeer BMP-4 Partial cDNA

The nucleotide sequence obtained from ABI Prism reactions was analyzed by computer

: · Ί; and compared with already known BMP sequences. New cloned cDNA

··· was found to be most homologous with deer BMP-4 (nucleotide homology 99% and amino acid homology 99%) and mouse, rat human, and canine BMP-4 (98% amino acid homology) based on homology comparisons. The nucleotide and amino acid homologues of BMP-4 proteins between different mammals are shown in · · ·. *. in Table 1.

Expression of the mature part of reindeer BMP-4 First, the mature part of reindeer BMP-4 was cloned into pTrcHis2A vector was transformed into E. coli TOP 10 cells to give the pTrcrd4 / 116 vector. Expression of the recombinant protein was induced by IPTG. Production of the recombinant protein was confirmed by SDS-119373 29 PAGE, but no induction was observed. This was thought to be due to many of the rdBMP-4 codons rarely used in E. coli. It is also possible that the inducibility is due to the high GC content at the beginning of the protein coding region of rdBMP-4 (GC-5 content of the first 10 codons is 70%).

For these reasons, it was decided to try a different vector system and a different E. coli strain. pET22b (+) (Novagen) with His6-tag and pelB leader region was selected as the new expression vector and Rosetta (DE3) (Novagen) and Origami B (DE3) 10 (Novagen) as new E. coli strains. The mature portion of reindeer BMP-4 was cloned into the pET22b (+) vector and the new plasmid was called pETrd4 and both Rosetta (DE3) and Origami B (DE3) cells were transformed with this vector. Analyzes by SDS-PAGE indicated that rdBMP-4 protein was overexpressed. Based on expression experiments, mainly Rosetta (DE3) cells containing the pETrd4 vector were used to produce the rdBMP-4 protein.

Purification of rdBMP-4

Reindeer rdBMP-4 recombinant protein was overexpressed in E. coli. After washing, precipitation based on 20 isoelectric points and solubilization of the inclusion bodies, the recombinant reindeer had a rdBMP-4 content of 85%.

• · • · · · ·

The next purification step was immobilized metal affinity chromatography (IMAC). T | The sample was eluted from the column using a pH gradient and the rdBMP-4 protein had a purity of at least 75% as determined by SDS-PAGE (Figure 8). The mature portion of the isolated rdBMP-4 protein had a molecular weight of 17,800 Da as determined by SDS-PAGE under electrophoretic mobility under reducing conditions.

Folding and Activity Testing of rdBMP-4:: *: 30 • ·

The in vitro folding of the denatured rdBMP-4 protein was quantitated. *. by measuring the folded dimer on Coomassie Brilliant Blue stained gels • · «densitometrically. The folding of the protein · · * ··· 'from non-reducing SDS-PAGE gels was 50%.

* • * _ _ 35 ·: ··; The osteoinductive activity of rdBMP-4 increased with increasing dose size (Table 2). At all concentrations tested, the reindeer rdBMP-4 recombinant protein proved to be a much more potent inducer of bone formation than the human one.

The present invention has been described with emphasis on some preferred embodiments and embodiments. However, it will be apparent to those skilled in the art that variations on the embodiments disclosed may be made and practiced, and that the invention may be practiced within the scope of the following claims other than as specifically set forth herein.

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SEQUENCE LISTING

<110> BBS-Bioactive Bone Substitutes Oy 5 <120> Bone Morphogenetic Proteins <130> OP10087 6 <160> 1 10 <170> Patent Version 3.1 <210> 1 <211> 116

15 <212> PRT

<213> Rangifer tarand tarand <400> 1 20 Ser Pro Lys His His Pro Gin Arg Arg Lys Lys Asn Lys Asn Cys 15 10 15

Arg Arg His Ser Pro Tyr Or Asp Phe Ser Asp Val Gly Trp Asn Asp 20 25 30 25

Trp lie Val Ala Pro Pro Gly Tyr Gin Ala Phe Tyr Cys His Gly Asp 35 40 45

Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala lie 30 50 55 60

Val Gin Thr Leu Val Asn Ser Val Asn Ser Ser lie Pro Lys Ala Cys 65 70 75 80 I i: 35 Cys Val Pro Thr Glu Leu Ser Ala lie Ser Met Leu Tyr Leu Asp Glu .1 I 85 90 95 • ♦ · ♦ · ♦ · ♦ · ·. ·,: Tyr Asp Lys Val Val Leu Lys Asn Tyr Gin Glu Met Val Val Glu Gly ·. 1: 100 105 110: 40 * · 1 1 Cys Gly Cys Arg: 115 »« · ·••••••••••••••••••••••• • ♦ • • · * «« «« «

Claims (19)

119373 An isolated bone morphogenetic protein 4 (BMP-4), characterized in that it contains the consensus sequence P-Q-R-A / S-R / K-K / R-K / N / R / L-N / K-K / R / S-N / H / S-C- 2. The morphogenetic protein 4 av ben enligt patent krav 1, the translation protocol consensus sequence P-Q-R-A / S-R-K-K / R-N / K-K / R-N / H-C-R-R-H-S / A-P. Bone morphogenetic protein 4 according to claim 1, characterized in that it contains the consensus sequence P-Q-R-A / S-R-K-K / R-N / K-K / R-N / H-C-R-R-H-S / A-P. 10 3. Morphogenetic protein 4 av ben enligt patent krav 1, transcriptase 5 attehäller aminosyrorna 6-21 av SEQ ID NO: 1. Bone morphogenetic protein 4 according to claim 1, characterized in that it contains amino acids 6-21 of SEQ ID NO: 1. Morphogenetic protein 4 av ben enligt patent krav 1, transcriptase att att innehäller aminosyrorna 1-23 av SEQ ID NO: 1. The bone morphogenetic protein 4 according to claim 1, characterized in that it contains amino acids 1-23 of SEQ ID NO: 1. The morphogenetic protein 4 of the amino acid sequence of SEQ ID NO: 1. The bone morphogenetic protein 4 according to claim 1, characterized in that it contains the amino acid sequence of SEQ ID NO: 1. 5 R / K-R-H-S / A-P or C-R / K-R-H-S / A-P-Y-V-D-F-S-D. 6. Isolerad DNA molecule, encoding the morphogenetic protein av ben enligt face av patent craven 1-5. An isolated DNA molecule, characterized in that it encodes a bone morphogenetic protein according to any one of claims 1-5. • · • · * • · · · · 7. Nucleotide vector, coding sequence for isolation of DNA DNA molecule patent krav 6. Nucleotide vector, characterized in that it contains an isolated DNA molecule according to claim 6. 25 • · i *: **. ·. Recombinant host cell, characterized in that it contains the nucleotide vector of claim 7. • · • f ·. . Pharmaceutical composition, characterized in that it contains a bone morphogenetic protein according to any one of claims 1-5. • • • • • • *; The pharmaceutical composition according to claim 9, characterized by ··· ·. ·· *. that it contains the bone morphogenetic protein in question as a homodimer, or immediate, ·. as a rhodimer with another bone morphogenetic protein. • · · * · “35 8. Recombinant cell line, nucleotide nucleotide nucleic acid nucleic acid sequence Tor enligt patent krav 7. 9. Farmaceutisk sammansättning, kännetecknad av att den innehäller ett mor-. . dental gene protein enligt face av patentkraven 1-5. • «· • · · * · 10. The pharmaceutical composition of claim 9, the codenamed av att den innehäller introduces a morphogenetic protein av ben som homodimer, an eller som heterodimer protein medication to a morphogenetic protein av ben. • ·· · • • · · 11. Pharmaceutisk sammansättning enligt patent krav 9 eller 10, kangnetecknad av • · * ··· 1 att den dessutom innehäller to provide morphogenetic protein av ben, en epidermal tillväxtfaktor, en fibroblastisk tillväxtfaktor eller en transformerande tillväxtfaktor. · • · «• · · · · · ***. 12. Användning av morphogenetic protein av ben enligt face av patentkra- · · ·. 25 ven 1-5 vid framställning av et on the go, med vilket man Värdar störningar i ben, • · · 'll ·' brosk, leder eller tänder, colors regeneration, reparation eller tillväxt av dessa är • · * ··· 1 self-colored. ·· · • 1 · • · Pharmaceutical composition according to claim 9 or 10, characterized in that it further comprises another bone morphogenetic protein, an epidermal growth factor, a fibroblastic growth factor or a transforming growth factor. 'N 119373 Use of a bone morphogenetic protein according to any one of claims 1 to 5 in the manufacture of a medicament for the treatment of disorders associated with bone, cartilage, tendons or teeth, where regeneration, repair or growth is desirable. 13. Användning av morphogenetic protein av ben enligt face av patent craven 1-5 vid framställning av on the basis of medication, mankind, cancer, fibromy-30, psoriasis and andra dermatologic störningar, and rheumatic color. 119373 Use of a bone morphogenetic protein according to any one of claims 1 to 5 in the manufacture of a medicament for the treatment of cancer, fibromyalgia, psoriasis and other dermatological disorders, and rheumatic disorders. 10 14. An osteogenic agent, an anatomic protein, and a morphogenetic protein av ben enligt face av patent kraven 1-5. An osteogenic device, characterized in that it contains a bone morphogenetic protein according to any one of claims 1 to 5. 15. The osteogenic agent encompasses patent application 14, the coding protocol discloses a morphogenetic protein av ben som homodimer, an eller som heterodimer till the same morphogenetic protein av. An osteogenic device according to claim 14, characterized in that it contains said bone morphogenetic protein as a homodimer, or as a heterodimer with another bone morphogenetic protein. 16. An osteogentic medication patent 14 eller 15, a transcriptional protocol to provide a morphogenetic protein av ben, an epidermal till factor, a fibroblastic till factor, and a transform factor. An osteogenic device according to claim 14 or 15, characterized in that it further contains a bone morphogenetic protein, an epidermal growth factor, a fibroblastic growth factor or a transforming growth factor. 17. Osteogent medel enligt flashes som Heist av patent engraver 14-16, transcript-10 nat av att detehehler in a biologically compatible matrix. An osteogenic device according to any one of claims 14 to 16,. characterized in that it contains a biocompatible matrix. The osteogenic device according to claim 17, characterized in that said biocompatible matrix contains calcium phosphate, carboxymethyl cellulose or collagen or their combinations. • · · • · · A method of inducing bone, cartilage, tendon or tooth formation in vitro, characterized in that the bone morpho ··· genetic protein according to any one of claims 1 to 5 is used to treat said bone, cartilage, tendon or tooth. «· • · · • · · •. * ··. Patent Isolation: 1. Isolerat Morphogenetic Protein 4 (BMP-4) av ben, transcriptome att att: ··· 35 innehäller consensus sequence PQRA / SR / KK / RK / N / R / LN / KK / R / SN / H / SC- R / KRHS / AP eller CR / KRHS / APYVDFSD. 119373 18. Osteogent medel enligt patent krav 17, a translucent matrix innate calcium phosphate, carboxymethylcellulose eller collagen eller combiner. 19. Förfarande för att inducera in vitro ben, brosk, led eller tandbildning, kän-15 netecknat av attörda ben, brosk, led eller tand behandlas med morphogenetic protein av benent face av patent kraven 1-5. •••••••••••••••••••••••••••••••••••••••••••• · · · · · · · · · · · · · · · · ··· • · ··· ........ ·· · a · · • · • · 1