The invention provides fusion polypeptides comprising protein transduction domains and osteoinductive polypeptides, as well as methods of using such polypeptides to induce osteogenesis and to promote proteoglycan synthesis. The invention also provides osteoinductive peptides which have demonstrated the ability to induce bone formation in vivo.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention provides a method of producing a cell-permeable osteoinductive polypeptide comprising introducing into a suitable host cell an expression construct encoding a cell-permeable polypeptide and an osteoinductive polypeptide positioned so that the osteoinductive polypeptide is expressed as part of a fusion protein with the cell-permeable polypeptide. The expression construct generally contains a promoter positioned to direct transcription of the polynucleotide sequence encoding the fusion product.

The expression construct may further comprise a purification tag. The cell-permeable polypeptide may be chosen from the group consisting of HIV-TAT, VP-22, a growth factor signal peptide sequence, Pep-1, and a Drosophila Antp peptide. The osteoinductive polypeptide may be chosen from the group consisting of LMP-1, LMP-3, SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, BMP-2, BMP-4, BMP-6, BMP-7, TGF-beta 1 and Smad.

The invention provides osteoinductive polypeptides chosen from among the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, and SEQ ID NO 8, or combinations thereof.

The invention also provides a method of inducing bone formation in a mammal comprising administering an effective amount of a fusion polypeptide comprising a protein transduction domain and at least one osteoinductive polypeptide. The fusion polypeptide may be administered as an implant and may be administered to at least one multipotent progenitor cell, which can be implanted into a mammal to promote osteoinduction.

The invention also provides a polynucleotide encoding a fusion protein comprising a protein transduction domain and at least one osteoinductive polypeptide, the protein transduction domain being chosen from among a variety of protein transduction, membrane-translocation, and other similar polypeptides represented, for example, by HIV-TAT, VP-22, a growth factor signal peptide sequence, Pep-1, and a Drosophila Antp peptide. The osteoinductive polypeptide may be chosen from the group consisting of LMP-1, LMP-3, SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, BMP-2, BMP-4, BMP-6, BMP-7, TGF-beta 1 and Smad.

A method of inducing proteoglycan synthesis in a mammal is also provided. The method comprises administering an effective amount of a fusion polypeptide comprising a protein transduction domain and at least one osteoinductive polypeptide. The fusion polypeptide may be administered as an implant, and may be administered to at least one multipotent progenitor cell.

An isolated fusion polypeptide comprising a membrane-translocating peptide operably linked to an osteoinductive polypeptide is provided by the invention. The membrane-translocating peptide may be chosen from the group consisting of HIV-TAT, VP-22, a growth factor signal peptide sequence, Pep-1, and a Drosophila Antp peptide and the osteoinductive polypeptide may be chosen from the group consisting of LMP-1, LMP-3, SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, BMP-2, BMP-4, BMP-6, BMP-7, TGF-beta 1 and Smad.

The invention provides a method of inducing osteoblast differentiation in a progenitor cell, the method comprising administering to the progenitor cell an effective amount of a fusion polypeptide comprising a protein transduction domain and at least one osteoinductive polypeptide. The protein transduction domain can be chosen from the group represented by HIV-TAT, VP-22, a growth factor signal peptide sequence, Pep-1, and Drosophila Antp polypeptides and the osteoinductive polypeptide may be chosen from the group represented by LMP-1, LMP-3, SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, BMP-2, BMP-4, BMP-6, BMP-7, TGF-beta 1 and Smad.

DETAILED DESCRIPTION

The inventors have discovered that a fusion protein comprising a protein transduction polypeptide and an osteoinductive polypeptide can be effectively used to promote bone development and intervertebral disc regeneration in vivo. The invention therefore provides osteoinductive polypeptides for intracellular delivery, polynucleotides encoding such osteoinductive polypeptides and protein transduction sequences, and methods of utilizing these fusion proteins to promote bone development and intervertebral disc regeneration in vivo.

Previous work has demonstrated that LIM mineralization protein splice variants 1 and 3 (LMP-1 and LMP-3) are osteoinductive, while LMP-2 does not appear to have such osteoinductive potential. A forty amino acid sequence corresponding to amino acids 94-133 of the amino acid sequence of human LMP-1 (hLMP-1) is common to both LMP-1 and LMP-3. The inventors therefore surmised that this unique region of the proteins might, in itself, have osteoinductive potential. Peptides comprising overlapping segments of this sequence were designed and used to test the inventors' hypothesis. Their results indicate that peptides derived from LMP-1 and LMP-3 have osteoinductive potential. When used in vivo, these peptides demonstrated the ability to induce bone formation. FIG. 6 (see Original Patent) indicates peptides which have demonstrated osteoinductive functionality when introduced into cells as part of the fusion protein of the present invention in the method of the present invention.

Protein transduction polypeptides facilitate the uptake and subsequent expression of nucleic acid sequences or therapeutic proteins. In the literature, they may be referred to alternately, and often interchangeably, as cell-permeable peptides, protein transduction domains, membrane transport sequences, and membrane-translocating peptides. They function to transport an attached peptide, polypeptide, or protein through the cell membrane into the interior of the cell in a receptor-independent manner. A fusion protein utilizing a protein transduction domain can comprise one or more peptides, polypeptides, or proteins operably linked to the protein transduction domain. In the present invention, such a fusion protein can comprise a protein transduction domain and at least one osteoinductive peptide, polypeptide, or protein, or combinations thereof. These peptides can be used to transduce autologous, allogeneic, or xenogeneic cells or tissues of ectodermal, mesenchymal, or hematopoetic origin and infuse or implant them into the recipient to induce or contribute to the formation of new tissue. In the method of the present invention, such polypeptides facilitate the uptake of proteins that can induce cells such as, for example, multipotent progenitor (stem) cells, to produce, for example, BMP-2, BMP-4, BMP-6, BMP-7, BMP-9, BMP-12, BMP-13, aggrecan, collagen type I, collagen type II, versican, lumican, fibromodulin, biglycan, and decorin. Effective amounts of polypeptides of the present invention are indicated in the experimental design and results disclosed herein, but may also be determined by one of skill in the art based upon the disclosure of effective amounts provided herein.

Human LIM mineralization protein-1 (hLMP-1), one of a family of LMP proteins, is an intracellular regulatory protein that can enhance the efficacy of bone mineralization in vitro and in vivo. Human LMP-1 is so named because it possesses a characteristic structural motif composed of two special zinc fingers that are joined by an amino acid spacer. LIM mineralization protein splice variants and their uses have been described by the inventors in U.S. Pat. Nos. 6,300,127; 6,444,803; and 6,521,750. The sequences of LMP-1, LMP-2, and LMP-3 have also been disclosed in those patents. On Jul. 22, 1997, a sample of 10-4/RLMP (Rattus norvegicus LIM mineralization protein cDNA) in a vector designated pCMV2/RLMP (which is vector pRc/CMV2 with insert 10-4 clone/RLMP) was deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, and was assigned accession number 209153. On Mar. 19, 1998, a sample of the vector pHis-A with insert HLPM is (Homo sapiens LIM mineralization protein cDNA) was deposited at the American Type Culture Collection and assigned accession number 209698.

A serotype 5 adenovirus (Ad5) has been employed for the delivery of LMPs to a variety of cells and tissues including cells derived from peripheral blood and bone marrow. (Boden, et al., "Adenoviral Delivery of LMP-1 Induces Consistent Spine Fusion", 47.sup.th Annual Meeting, Orthopaedic Research Society, San Francisco, Calif. (2001)). However, the Ad5 virus utilizes a specific receptor (i.e., coxsackie adenovirus receptor or CAR), which is absent, or present in limited quantities, in these cells. Protein transduction across the cell membrane to facilitate intracellular delivery of proteins without receptor-mediated mechanisms offers an attractive alternative to allow treatment of a variety of cell and tissue types.

The actions of LMPs and other osteoinductive proteins indicate that they have therapeutic potential in a variety of tissues, such as brain, spinal cord, peripheral nerve, bone, cartilage, intervertebral discs, connective tissue, tendons, and ligaments. Delivery of LMPs, for example, to a variety of tissues can be accomplished by delivery systems comprising, for example, collagen, collagen ceramic combinations, demineralized bone matrix, natural or synthetic polymers such as elastin, fibrin, polylactic acid, polyglycolic acid, polycaprolactone, polypropylene fumarate, polyvinyl alcohol, polyesters, polyethers, polyhydroxyls, and structural implants. Such matrices may be injectable, moldable, solid implants, structural implants, or combinations thereof.

The present inventors have discovered that PTDs can be used to deliver functional osteoinductive proteins into cells and to effectively induce osteogenesis and proteoglycan synthesis. Such cell-permeable peptide import (CPPI) provides a method for delivering osteoinductive proteins into a variety of cell types. An 11 amino acid peptide, initially derived from the HIV-1 TAT protein, was successfully used to deliver osteoinductive proteins into cells. The TAT peptide can be over-expressed in bacterial cells using the pTAT-HA vector. A recombinant human gene can be inserted into this vector in such a manner as to produce a fusion protein containing both the TAT peptide sequence as well as the gene product of interest. Furthermore, the PTD/osteoinductive polypeptide can be expressed in conjunction with a polyHis tag in order to facilitate isolation and purification of the fusion protein. The pTAT-HA vector and a purification protocol for TAT fusion proteins have been described previously by Nagahara, et al. (Nature Medicine, Vol. 4) p. 1449-1452, December 1998).

A peptide sequence as found in a variety of PTDs can facilitate entry into cells in a coxsackie-adenovirus receptor (CAR)-independent manner, thereby improving transduction efficiencies to target cells and subsequently lowering the required amounts of nucleic acid or protein needed to achieve the desired effect. PTD fusion proteins therefore provide a therapeutic tool that may be used to reduce the cost of therapy.

In one embodiment of the invention, a fusion protein of a protein transduction domain and an osteoinductive protein is provided. Osteoinductive proteins include, but are not limited to, LIM mineralization proteins (LMPs), bone morphogenetic proteins (BMP) and Smad proteins. As used herein, "osteoinductive proteins," "osteoinductive polypeptides," and "osteoinductive peptides" may be used interchangeably to refer to either a peptide or polypeptide of varying length or a full-length protein with osteoinductive functionality.

A fusion protein comprising a PTD and a LIM mineralization protein is provided as one embodiment of the invention. The fusion protein can comprise a PTD and one or more LIM mineralization proteins or polypeptides. Useful LIM mineralization proteins include, for example, LMPs as disclosed in U.S. Pat. Nos. 6,300,127; 6,444,803; and 6,521,750; as well as pending U.S. patent application Ser. No. 09/959,578, filed Apr. 28, 2000. Preferably, the LMP is RLMP, HLMP-1, HLMP-1s, HLMP-2, HLMP-3, or a peptide derived therefrom. These peptides can include, for example, SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, or a polypeptide as in SEQ ID NO 8.

The nucleotide sequence encoding the LIM mineralization protein preferably hybridizes under standard conditions to a nucleic acid molecule complementary to the full length of the following sequence:

tcctcatccg ggtcttgcat gaactcggtg (SEQ. ID. NO. 9)

or hybridizes under highly stringent conditions to a nucleic acid molecule complementary to the full length of the following sequence:

gcccccgccc gctgacagcg ccccgcaa (SEQ. ID. NO. 10),

or both.

"Standard hybridization conditions" will vary with the size of the probe, the background and the concentration of the nucleic acid reagents, as well as the type of hybridization (in situ, Southern blot, or hybridization of DNA-RNA hybrids (Northern blot)). The determination of "standard hybridization conditions" is within the level of skill in the art. Methods include, for example, those described in U.S. Pat. No. 5,580,775 (Fremeau, et al.), Southern, J. Mol. Biol., 98:503 (1975), Alwine, et al., Meth. Enzymol., 68:220 (1979), and Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, 7.19-7.50 (1989).

One set of standard hybridization conditions involves pre-hybridizing a blot at 42.degree. C. for 2 hours in 50% formamide, 5.times.SSPE (150 nM NaCl, 10 mM NaH.sub.2PO.sub.4 [pH 7.4], 1 mM EDTA [pH 8.0]) 5.times.Denhardt's solution (20 mg Ficoll, 20 mg polyvinylpyrrolidone and 20 mg BSA per 100 ml water), 10% dextran sulphate, 1% SDS and 100 .mu.g/ml salmon sperm DNA. A .sup.32P-labeled cDNA probe is added, and further hybridizing continued for 14 hours. Afterward, the blot is washed twice with 2.times.SSPE, 0.1% SDS for 20 minutes at 22.degree. C., followed by a 1 hour wash at 65.degree. C. in 0.1.times.SSPE, 0.1% SDS. The blot is then dried and exposed to x-ray film for 5 days in the presence of an intensifying screen.

Under "highly stringent conditions", a probe will hybridize to its target sequence if those two sequences are substantially identical. Techniques are known to those of skill in the art for determining the conditions under which only substantially identical sequences will hybridize while non-identical sequences will not.

As used herein, the term "protein" is intended to include mimetics and the term "amino acid" is intended to include L-form, D-form, and modified amino acids. These substitutions may be made by one of skill in the art, using the known structural similarities between the molecules. The amino acid sequence is also intended to include any peptide or protein sequence that may include additional amino acids either N-terminal or C-terminal to the listed sequence, or both. The term "osteoinductive protein" is intended to include variants or biologically active fragments of the polypeptide, as well as full-length proteins.

It is well known in the art that a single amino acid may be encoded by more than one nucleotide codon, and that the nucleotide sequence may be modified to produce an alternate nucleotide sequence that encodes the same peptide. Therefore, alternate embodiments of the present invention include alternate DNA sequences encoding peptides containing the amino acid sequences as previously described. DNA sequences encoding peptides containing the claimed amino acid sequence include DNA sequences which encode any combination of the claimed sequence and other amino acids located N-terminal or C-terminal to the claimed amino acid sequence. It is to be understood that amino acid and nucleic acid sequences may include additional residues, particularly N- or C-terminal amino acids or 5' or 3' nucleotide sequences, and still be essentially as set forth in the sequences disclosed herein, as long as the sequence confers osteoinductive potential upon the expressed polypeptide or protein.

Additional nucleic acid bases may be added either 5' or 3' to the nucleotide sequence encoding the osteoinductive polypeptide, and may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like. Therefore, overall length of such a polynucleotide may vary considerably.

It is to be understood that a "variant" of a polypeptide is not completely identical to the native protein. A variant of an osteoinductive polypeptide or protein, for example, can be obtained by altering the amino acid sequence by insertion, deletion or substitution of one or more amino acids. The amino acid sequence of the polypeptide or protein can be modified, for example, by substitution to create a polypeptide having substantially the same or improved qualities as compared to the native polypeptide. The substitution may be a conserved substitution. A "conserved substitution" is a substitution of an amino acid with another amino acid having a side chain that is similar in polar/nonpolar nature, charge, or size. The 20 essential amino acids can be grouped as those having nonpolar side chains (alanine, valine, leucine, isoleucine, proline, phenylalanine, and tryptophan), uncharged polar side chains (methionine, glycine, serine, threonine, cystine, tyrosine, asparagine and glutamine), acidic side chains (aspartate and glutamate), and basic side chains (lysine, arginine, and histidine). Conserved substitutions might include, for example, Asp to Glu, Asn, or Gln; His to Lys, Arg or Phe; Asn to Gln, Asp or Glu; and Ser to Cys, Thr or Gly. Alanine, for example, is often used to make conserved substitutions.

To those of skill in the art, variant polypeptides can be obtained by substituting a first amino acid for a second amino acid at one or more positions in the polypeptide structure in order to affect biological activity. Amino acid substitutions may, for example, induce conformational changes in a polypeptide that result in increased biological activity.

Those of skill in the art may also make substitutions in the amino acid sequence based on the hydrophilicity index or hydropathic index of the amino acids. A variant amino acid molecule of the present invention, therefore, has less than one hundred percent, but at least about fifty percent, and preferably at least about eighty to about ninety percent amino acid sequence homology or identity to the amino acid sequence of a polypeptide comprising the amino acid sequence of LMP-1, LMP-2, LMP-3, SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, or a polypeptide as in SEQ ID NO 8. Therefore, the amino acid sequence of the variant osteoinductive polypeptide or protein corresponds essentially to the native osteoinductive polypeptide or protein amino acid sequence. As used herein, "corresponds essentially to" refers to a polypeptide sequence that will elicit a similar biological and enzymatic activity to that generated by an osteoinductive polypeptide or protein comprising LMP-1, LMP-2, LMP-3, SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, or a polypeptide as in SEQ ID NO 8, such activity being at least about 70 percent that of the native osteoinductive protein, and more preferably greater than 100 percent of the activity of the native osteoinductive protein.

A variant of the osteoinductive protein may include amino acid residues not present in a corresponding osteoinductive protein comprising LMP-1, LMP-2, LMP-3, SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, or SEQ ID NO 8, or may include deletions relative to the osteoinductive protein comprising LMP-1, LMP-2, LMP-3, SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, or SEQ ID NO 8. A variant may also be a truncated "fragment," as compared to the corresponding protein comprising LMP-1, LMP-2, LMP-3, SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, or SEQ ID NO 8, the fragment being only a portion of the full-length protein or polypeptide.

Bone morphogenetic proteins (BMPs) are members of the TGF-.beta. superfamily of proteins. BMPs have been shown to induce ectopic bone or cartilage formation. According to the invention, a fusion protein of a PTD and a bone morphogenetic protein is also provided. BMPs include, for example, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, GDF-1, GDF-3, GDF-8 and GDF-9. Bone morphogenetic proteins BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, or BMP-9 can be especially useful in the method of the present invention.

Smad proteins are intracellular proteins that mediate signaling from receptors for extracellular TGF-beta-related factors (Heldin. et al., "TGF-.beta. Signalling from Cell Membrane to Nucleus through SMAD Proteins", Nature, Vol. 390 (1997)). Smad proteins can be activated (i.e., phosphorylated) by the binding of a BMP to its receptor. Upon activation, the Smad proteins translocate to the nucleus where they regulate gene expression. A fusion protein of a PTD and a Smad protein is also provided in the present invention. Smad-1, Smad-2, Smad-3, Smad-4, Smad-5, Smad-6, Smad-7 or Smad-8 can be especially useful for promoting osteoinduction when delivered as a fusion protein with a protein transduction domain as in the present invention.

The protein transduction domain according to the invention can be any peptide, mimetic, or peptide nucleic acid (PNA) sequence that can traverse the plasma membrane of a cell to deliver an attached or accompanying protein, peptide, or nucleic acid to the interior of the cell. The inventors have demonstrated that osteoinductive proteins can be delivered intracellularly (as a fusion protein moiety, for example) without impairing their ability to promote osteoinduction and proteoglycan synthesis. PTDs include, for example, polypeptides derived from the Drosophila homeotic transcription factor Antennapedia (Antp), the herpes simplex virus (HSV) protein VP22, signal peptide sequences from growth factors such as Kaposi's fibroblast growth factor (K-FGF) (Lin, et al., J. Biol. Chem., Vol. 270, p. 14255-14258, 1995) a membrane translocation sequence derived from the K-FGF signal peptide sequence (Rojas, et al, Nat. Biotech., Vol. 16, p. 370-375, 1998), and the human immunodeficiency virus (HIV)-1 transcriptional activator TAT (Fawell, et al., Proc. Natl. Acad. Sci. USA, Vol. 91, p. 664-668, 1994). PTDs are disclosed in U.S. Pat. No. 5,652,122, and in Schwarze. et al., "Protein Transduction: Unrestricted Delivery into all Cells", Trends in Cell Biology, Vol. 10 (2000). The inventors have found the HIV-TAT PTD to be especially useful in the present invention.

A nucleic acid comprising a nucleotide sequence encoding a fusion protein operably linked to a promoter, wherein the fusion protein comprises a protein transduction domain (PTD) and an osteoinductive protein, is also provided. The nucleic acid can be part of a vector (e.g., an expression vector such as a plasmid). Osteoinductive proteins can include, for example, LIM mineralization proteins, bone morphogenetic proteins, Smad proteins, and osteoinductive peptides and polypeptides derived therefrom. Examples of osteoinductive peptides and polypeptides include SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, and SEQ ID NO 8.

Methods of delivering osteoinductive proteins into cells are also provided by the present invention. In a method of the invention, at least one osteoinductive protein can be delivered into a cell via transduction wherein a fusion protein comprising a protein transduction domain (PTD) and an osteoinductive protein is contacted with the cell so that the fusion protein is delivered into the cell, the delivery being facilitated by the protein transduction domain or cell-permeable peptide

The cells into which the osteoinductive proteins can be delivered include, for example, osseous (i.e., bone forming) and non-osseous cells. Such cells may include, for example, buffy coat cells, stem cells (e.g., mesenchymal stem cells, multipotent and pluripotent stem cells), intervertebral disc cells (e.g., cells of the annulus fibrosus and cells of the nucleus pulposus), mesenchymal cells, hematopoietic cells, endothelial cells and muscle cells. Stem cells can be derived from autalogous or allogeneic tissue.

Cells transduced with or expressing a fusion protein of a protein transduction domain (PTD) and an osteoinductive protein are also provided. Such cells may include, but are not limited to, buffy coat cells, stem cells (e.g., mesenchymal stem cells and pluripotential stem cells), intervertebral disc cells (e.g., cells of the annulus fibrosus and cells of the nucleus pulposus), mesenchymal cells, hematopoietic cells, endothelial cells and muscle cells. Cells containing a fusion protein of a PTD and an osteoinductive protein as described herein can be implanted into the body of a mammal to induce bone formation. Methods of inducing bone formation using LMPs as osteoinductive proteins are described, for example, in U.S. Pat. No. 6,300,127. Cells comprising a fusion protein of a PTD and an osteoinductive protein may also be implanted into the intervertebral disc, for example, to stimulate proteoglycan and/or collagen synthesis as set forth in U.S. patent application Ser. No. 10/292,951, filed Nov. 13, 2002, pending.

A Conjugate of a PTD and a nucleic acid comprising a nucleotide sequence encoding an osteoinductive protein is also provided. The PTD/nucleic acid conjugate can be used to direct over-expression of an osteoinductive protein to promote bone formation or disc regeneration, for example. Osteoinductive proteins encoded by the nucleotide sequence can include, but are not limited to, LMPs, BMPs, and Smad proteins. Methods for chemically linking peptides to nucleic acids are known in the art. One such method is described in U.S. Pat. No. 5,652,122. The nucleic acid can be in the form of an expression vector comprising a nucleotide sequence encoding an osteoinductive protein operably linked to a promoter.

Methods of the present invention can be used to induce the expression of one or more bone morphogenetic proteins or transforming growth factor-.beta. proteins in a cell as described in copending U.S. patent application Ser. No. 10/382,844, filed Mar. 7, 2003. For example, the expression of one or more proteins selected from the group consisting of BMP-2, BMP-4, BMP-6, BMP-7, TGF-.beta.1 and combinations thereof can be induced by contacting a cell with a fusion protein comprising a PTD and an osteoinductive protein according to the invention. Additionally, cells which over-express one or more proteins selected from the group consisting of BMP-2, BMP4, BMP-6, BMP-7, TGF-.beta.1 and combinations thereof are also provided according to the invention. The cell can be any somatic cell including, but not limited to, a stem cell, a buffy coat cell, a bone marrow cell, a peripheral blood cell or a fat cell. The cell can be a stem cell derived from autologous or allogeneic tissue.

Stem cells, or multipotent progenitor cells, can provide a source of cells from which to generate osteoblasts. These cells may be isolated at various stages of differentiation and induced to differentiate in specific lineage pathways. The cells may be used to treat bone diseases such as osteoporosis or osteogenesis imperfecta, as well as non-healing fractures. Core binding factor alpha 1 (Cbfa1) has been demonstrated to be necessary for osteogenesis. BMP-2, BMP-4, and BMP-7, which are known to induce osteoblast differentiation, up-regulate Cbfa1 expression. BMP-8 and Smad-3 are up-regulated during osteoblast differentiation. Activation of TGF-beta/BMP-Smad signaling has been shown to promote Cbfa1 expression, and osteoblast differentiation. The present invention provides fusion proteins comprising functional BMPs, LMPs, Smad proteins, or a combination thereof, for example, to promote osteoblast differentiation in cells such as human bone marrow-derived mesodermal progenitor cells. Suitable cells may include, for example, multipotent cells such as those described by Jiang, et al. (Nature, Vol. 418, p. 41-49, 2002). Administration of suitable osteoinductive proteins or polypeptides, or combinations thereof, can be performed ex vivo before implantation of the cells, or in vivo following implantation or injection. For in vivo administration, osteoinductive proteins of the present invention can be injected at a target site so that they can be delivered to the interior of nearby cells via a PTD or cell-permeable peptide, for example. Alternately, an implant comprising a carrier in combination with a PTD/osteoinductive polypeptide may be used. Implants may contain reservoirs in which to place the PTD/osteoinductive polypeptide for release into the surrounding tissue, or may comprise a porous composition that has been soaked in a solution containing one or more PTD/osteoinductive polypeptide constructs. Hydrogels, time-release capsules or spheres, liposomes, microspheres, nanospheres, biodegradable polymers, or other such drug delivery systems may also be employed to deliver peptides and proteins of the present invention to target cells and tissues. U.S. Pat. No. 6,475,516 (DiCosmo, et al.), for example, provides hydrogels loaded with liposomal therapeutic agents such as antibiotics, the hydrogels being covalently bonded to the surface of an in-dwelling medical device such as an implant.

A hallmark of disc degeneration is the decreased production of proteoglycans in the disc, especially sulfated-glycosaminoglycans (sGAG) and aggrecan. A decrease in the production rate of aggrecan, the major proteoglycan of the intervertebral disc, is an important factor in intervertebral disc degeneration. Because of the central role of proteoglycans in the function of the intervertebral disc, restoration of normal proteoglycan production of the intervertebral disc may be critically important in any biological treatment of intervertebral disc degeneration.

The inventors performed experiments which demonstrated that LMP-1 over-expression or intracellular administration increases disc cell proteoglycan production in vitro and in vivo. LMP-1 over-expression induces the upregulation of BMP-2 and BMP-7 mRNA in vitro and in vivo. Noggin, which specifically inhibits these BMP-2 and BMP-7, inhibits proteoglycan upregulation by AdLMP-1, indicating that LMP-1 induced upregulation of proteoglycan is mediated by the upregulation of BMPs. LMP-1 administration via gene therapy or protein therapy (e.g., delivery by PTD conjugates) therefore can be used to stimulate proteoglycan production in discs and play a therapeutic role in disc regeneration.

Cytokines such as TGF-.beta.1, IGF-1, and EGF have been shown to stimulate intervertebral disc cell mitosis and, to some extent, proteoglycan production. Other cytokines such as BMP-2 and BMP-7 have also been shown to be effective in stimulating proteoglycan production. Because cytokines are small water soluble molecules, however, they rapidly diffuse away from the intervertebral disc or become inactivated by other regulatory factors. LIM Mineralization Protein-1 (LMP-1) is an intracellular regulatory molecule that is known to induce the secretion of multiple different BMPs from leukocytes and osteoblasts. By delivering LMP-1, LMP-2, LMP-3, or an osteoinductive peptide derived from LMP-1 or LMP-3, or a combination thereof, into the cell, particularly via a PTD/nucleic acid conjugate, BMP production can be stimulated from within the cells. Suitable osteoinductive peptides include, for example, SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, or a polypeptide as in SEQ ID NO 8.

Claim 1 of 22 Claims

1. A method of inducing bone formation in a mammal comprising administering an effective amount of a fusion polypeptide consisting of a protein transduction domain and an amino acid sequence selected from the group consisting of the amino acid sequence consisting of SEQ ID NO 1, the amino acid sequence consisting of SEQ ID NO 2, the amino acid sequence consisting of SEQ ID NO 4, the amino acid sequence consisting of SEQ ID NO 7, and the amino acid sequence consisting of SEQ ID NO 8.
 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.