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Intracellular delivery of osteoinductive proteins and peptides

This application claims the benefit of U.S. provisional application No. 60/456,551, filed Mar. 24, 2003.

The present invention relates generally to osteoinductive proteins and methods for the delivery of those proteins into cells. More specifically, the invention relates to osteoinductive proteins such as LIM mineralization proteins (LMPs), bone morphogenetic proteins (BMPs) and Smad proteins, conjugates of such osteoinductive proteins with protein transduction domains (PTDs), conjugates of PTDs and nucleic acids comprising nucleotide sequences encoding osteoinductive proteins, and to the transduction of these conjugates into cells. Furthermore, the invention relates to the use of PTD/osteoinductive protein conjugates to promote bone growth and disc regeneration.

Tissue regeneration is an important component of the healing process subsequent to disease, trauma, or surgery. In situations where disease or trauma produces bone defect, for example, or where a surgical procedure such as insertion of an autograft or allograft, bone bridge, or bone fusion is used to correct a bone defect, bone regeneration is a central goal of recovery. It is not, however, a goal that is always or easily achieved and much research has been devoted to newer and more effective ways to promote tissue repair and regeneration.

Elimination of joint motion by creation of a bone bridge is a common orthopedic strategy for the treatment of degenerative spine and joint disorders. Failure of spine fusion can occur in as many as forty-five percent of the patients who undergo the procedure, leaving them with continued pain, repeated surgeries, medical costs, and overall therapeutic failure.

Intracellular and extracellular osteoinductive proteins promote bone growth and repair and constitute potential targets for therapeutic use. Such proteins include the bone morphogenetic proteins and the LIM mineralization proteins. BMPs have been shown to stimulate bone growth in vivo and LMPs, particularly LMP-1 and LMP-3, have a more upstream effect on osteoinduction, as evidenced by the fact that inhibition of LMP-1 expression blocks nodule formation that would normally be stimulated by glucocorticoids or BMP-6. Since they are considered “extracellular” proteins, acting via interaction with cell surface receptors, very high doses of bone morphogenetic proteins are required to achieve consistent effects in humans. Since the manufacturing costs of BMPs is generally high, this can mean that the cost of therapy is prohibitive. Therefore, although BMPs have demonstrated efficacy and are a viable therapeutic aid to osteoinduction, it would be beneficial to develop an alternate therapy that might be more cost-effective and possibly even more therapeutically effective.

Delivery of LMPs to the intracellular environment provides an attractive therapeutic regimen. This can be accomplished by transfection of cells with plasmids comprising a nucleotide sequence encoding a LIM mineralization protein, or can be done by infection of target cells with a viral vector carrying the nucleotide sequence of LMP. Each of these techniques has limitations, however. Plasmid transfection generally requires that cells be isolated for transfection and then implanted after transfection. Viral delivery generally requires that the appropriate receptor be located on the surface of the target cell in order to facilitate viral entry into the cell.

There is tremendous potential for the use of osteoinductive proteins and peptides, particularly for the use of those proteins and peptides that act via an intracellular mechanism. What is needed is a method of delivery of effective intracellular osteoinductive proteins and peptides into cells.

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.