KR20070072938A - Intracellular delivery of osteoinductive proteins and peptides - Google Patents

Abstract

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

Intracellular Delivery of Osteoinductive Proteins and Peptides

The present invention relates to osteoinductive proteins and methods of intracellular delivery of such proteins. In particular, the present invention relates to nucleic acids comprising nucleotide chains encoding LIM degrading proteins (LMPs), bone marrow morphogenic proteins (BMPs) and Smad proteins, conjugates of osteoinductive proteins including protein delivery domains (PTDs), and osteoinductive proteins. And conjugates of PTDs, and intracellular delivery of such conjugates. Moreover, the present invention is directed to improving bone growth and disc regeneration using PTD / osinduced protein conjugates.

Tissue regeneration is an important component of the treatment process associated with disease, disorder, or surgery. Bone regeneration occurs when a disease or disorder results in bone defects, for example, surgical procedures such as the insertion of autografts or allografts, or correction of bone bonding using bone fusion. Is an important issue. However, this goal is not easily achieved, and many studies have been conducted to improve the treatment and regeneration of tissues.

Elimination of joint movement by bone bridges is a common orthopedic treatment for the treatment of degenerative spinal and joint diseases. Failed vertebral fusion occurs in 45 percent of surgical patients, causing them to continue to suffer and repeat surgery, resulting in overall medical failure due to increased medical bills.

Intracellular and extracellular bone-induced proteins enhance and treat bone growth, forming potential targets for therapeutic use. Such proteins include bone morphogenic proteins and LIM degrading proteins. BMPs have been shown to stimulate bone growth in vivo, in particular LMP-1 and LMP-3 have a significant effect on bone induction. Evidence for this is the inhibition of the formation of LMP-1 expressing block nodules, which are usually stimulated by glucocorticoids or BMP-6. Because they are considered "extracellular" proteins that interact with cell surface receptors, sustained effects can be achieved by overdosing bone morphogenic proteins. Since the manufacturing cost of BMPs is very high, this leads to an increase in the treatment cost. Therefore, although BMPs can be effective and useful in the treatment of bone induction, there is a need to develop alternative treatments that can be more cost-effective and further improve the therapeutic effect.

Delivery of LMPs to the intercellular environment provides an attractive treatment. This can be accomplished by cell transfection with a plasmid comprising nucleotide chains encoding LIM degradation proteins, or by infection of target cells with viral vectors carrying the nucleotide chains of LMP. However, this technique is extremely limited. Plasmid transfection should be transplanted by separating the cells from the transfection. Viral delivery should place the appropriate receptor on the surface of the target cell so that the virus can easily penetrate the cell.

The use of bone-derived proteins and peptides has great potential, especially how to activate them through intercellular mechanisms. What is needed is the transfer of effective intercellular bone inducing proteins and peptides to cells.

The present invention provides a method of making a cell-permeable osteoinductive polypeptide comprising positioning an osteoinductive polypeptide and an expression construct encoding a cell-permeable polypeptide in a suitable host cell so that the osteoinductive polypeptide is expressed as a fusion protein with the cell-permeable polypeptide. to provide. Expression constructs generally include a promoter positioned for direct transcription of the polynucleotide chain encoding the fusion product.

Expression constructs may also include purified tags. The cell permeable polypeptide may be selected from the group consisting of HIV-TAT, VP-22, growth factor signal peptide chain, Pep-1, and Drosophila Antp peptide. Osteoinduced polypeptides include 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 present invention is a 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, SEQ ID NO 8, or a mixture thereof Provided is an osteoinductive polypeptide selected from.

The present 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 or may be administered to at least one multipotent progenitor cell to be implanted in a mammal to enhance bone induction.

The invention also provides a polynucleotide encoding a fusion protein comprising a protein transduction domain and at least one osteoinductive polypeptide, wherein the protein transduction domain is comprised of several types of protein transduction, membrane translocation, and HIV-TAT, VP-22. , Growth factor signal peptide chain, Pep-1, and other similar proteins expressed as Drosophila Antp peptide. Osteoinduced polypeptides include 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.

Provided are methods for inducing proteoglycan synthesis in a mammal. 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.

Isolated fusion polypeptides include mebrain translocation peptides functionally associated with osteoinductive polypeptides, which are provided by the present invention. The membrane translocation peptide can be selected from the group consisting of HIV-TAT, VP-22, growth factor signal peptide chain, Pep-1, and Drosophila Antp peptide, and the osteoinductive polypeptide is 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 present invention provides a method of inducing osteoblast differentiation in progenitor cells, the method comprising administering to a progenitor cell an effective amount of a fusion polypeptide comprising a protein transduction domain and at least one osteoinductive polypeptide. The protein transduction domain is selected from the group represented by HIV-TAT, VP-22, growth factor signal peptide chain, Pep-1, and Drosophila Antp peptide, and the osteoinductive polypeptides are 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.

It has been found by the inventors that fusion proteins, including protein transducing polypeptides and osteoinductive polypeptides, can be effectively used to enhance bone growth and regeneration of the intervertebral discs in vivo. Accordingly, the present invention provides osteoinductive polypeptides for intercellular migration, polynucleotides and protein conversion chains encoding such osteoinductive polypeptides, and utilize the fusion proteins to enhance bone growth and regeneration of the intervertebral discs in vivo. Provide a method.

The prior art has shown that LIM degraded protein slice variants 1 and 3 (LMP-1 and LMP-3) are osteoinductive, whereas LMP-2 is not osteoinductive. The 40 amino acids corresponding to amino acids 94-133 of the amino acid chain of human LMP-1 (hLMP-1) are common to both LMP-1 and LMP-3. Thus, according to the present inventors, it can be summarized that the specific region of such a protein itself has osteoinduction. To test our hypothesis, peptides comprising overlapping segments of this chain are constructed and used. These results indicate that the peptides derived from LMP-1 and LMP-3 have osteoinductive properties. When used in vivo, these peptides exhibit the ability to induce bone formation. Figure 6 shows the osteoinductive function when the fusion protein of the present invention is injected into cells in the method of the present invention.

A method for implanting cell expressing LMP-1 into the thoracic or lumbar spine to form hard spinal fusion in amic mice has been shown by the inventors. Through the results shown here, LMP-1 such as 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 And LMP-3 peptides are also known to provide the osteoinductive effects shown in LMP-1.

Bone morphogenic proteins are generally in high doses and can maintain a lasting effect on the human body. LMP-1 intercellular migration by PTD fusion proteins as described above provides a more cost effective treatment. Moreover, the data results show that LMP-1 and LMP-related osteoinductive peptides act to cause factors to flow in cells to fulfill the natural osteoinductive pathway.

Protein transducing polypeptides facilitate the expression of nucleic acid chains or therapeutic proteins. In theory, they can be interchanged and replaced as cell penetrating peptides, protein transduction domains, membrane delivery chains, and membrane translocation peptides. They function to deliver the bound peptides, polypeptides or proteins intracellularly through a cell membrane in a receptor independent manner. Fusion proteins utilizing a protein translation domain may comprise one or more peptides, polypeptides or proteins functionally linked with the protein translation domain. In the present invention, such fusion proteins comprise a protein transduction domain and at least one osteoinductive peptide, polypeptide or protein, or mixtures thereof. These peptides can be used to transform autologous, allograft or xenograft cells or tissues of ectoderm, mesenchymal and hematopoietic tissues and can be used to form new tissues by injecting or transplanting them into receptors. In the methods of the invention, such polypeptides are selected from BMP-2, BMP_4, BMP-6, BMP-7, BMP-9, BMP-12, BMP-13, Agrecan, Collagen I, Collagen II, Versican, It facilitates the acquisition of proteins that can induce cells, such as multipotent progenitor (stem) cells, to produce lumicans, fibromodulin, aglycans and decorin. Although the effective amount polypeptides of the invention are shown experimentally and show results, they may also be determined by one skilled in the art based on the disclosed effective amounts.

Human LIM degrading protein-1 (hLMP-1), one of a group of LMP proteins, is an intercellular regulatory protein capable of enhancing osteolytic efficacy in vitro and in vivo. Human LMP-1 has a characteristic structural motif consisting of two specific zinc fingers bound by amino acid spacers. LIM degraded protein slice variants and their use are described in US Pat. Nos. 6,300,127, 6,444,803, and 6,521,750 by the inventors. LMP-1, LMP-2 and LMP-3 are also disclosed in this patent. On July 22, 1997, 10-4 / RLMP (Rattus norvegicus LIM Degrading Protein cDNA) in vector-designed pCMV2 / RLMP (vector pRc / CMV2 with insert 10-4 clone / RLMP) was produced by the American Type Culture Collection (ATTC). (12301 Parklawn Drive, Rockville, Md. 20852) and was assigned accession number 209153. On March 19, 1998, vector pHis-A (Homosapiens LIM Degrading Protein cDNA) containing insert HLMP-1 was deposited in the same institution and was assigned Accession No. 209698.

The serotype 5 adenovirus (Ad5) is applied to deliver LMPs to several cells and tissues, including cells derived from peripheral blood and bone grafts (Boden, et al., “Adenoviral Delivery of LMP-1 Induces Consistent Spine). Fusion ", 47th Annual Meeting, Orthopaedic Research Society, San Francisco California, 2001). However, Ad5 viruses use specific receptors (eg, coxsackie adenovirus receptors or CARs) that are absent or extremely limited in these cells. Protein conversion across cell membranes, which facilitates intercellular transport of proteins without mediating receptors, suggests an attractive choice, allowing the treatment of multiple cell and tissue types.

The activity of LMPs and other osteoinductive proteins has shown that they can be used to treat a variety of tissues such as brain, spinal cord, peripheral nerves, bone, cartilage, intervertebral discs, connective tissue, tendons and ligaments. For example, the migration of LMPs to various tissues can be characterized by collagen, collagen ceramic combinations, undigested bone matrix, elastin, fibrin, polyratic acid, polyglycolic acid, polycaprolactone, polypropylene fumarate as natural or synthetic polymers. , Polyvinyl alcohol, polyester, polyether, polyhydroxyl, and structural implants. Such matrices are by injection, molding, hard implants, structural implants or combinations thereof.

It has been found by the inventors that PTDs can be used to transfer functional osteoinduced proteins into cells and effectively induce osteoinduction and proteoglycan synthesis. Such cell permeable peptide import (CPPI) provides a method of transporting osteoinductive proteins to various types of cells. Eleven amino acid peptides originally derived from HIV-1 TAT protein have been used successfully to transfer osteoinductive proteins into cells. TAT peptides can be overexpressed in bacterial cells using the pTAT-HA vector. Recombinant human genes can be inserted into this vector to produce a fusion protein containing the TAT peptide chain and certain genetic material. Moreover, PTD / oskeletal induction polypeptides can be expressed covalently with a polyHis tag to facilitate purification and isolation of the fusion protein. PTAT-HA vectors and purification protocols for TAT fusion proteins have already been described by Nagahara et al. (Nature Medicine, Vol. 4, p. 1449-1452, December 1998).

Peptide chains found in several PTDs can be readily injected into cells CAR-independently to enhance the transformation of target cells and thus lower the amount of nucleic acid or protein needed to achieve the desired effect. Thus, PTD fusion proteins provide a therapy that can reduce the cost of treatment.

In one embodiment of the invention, a fusion protein of a protein transduction domain and an osteoinductive protein is provided. Osteoinduced proteins include LIM degrading proteins (LMPs), bone morphogenic proteins (BMP) and Smad proteins. The "bone guide proteins", "bone guide polypeptides" and "bone guide peptides" can be used differently to refer to peptides or polypeptides of different lengths, or full length proteins with osteoinductive efficacy.

Fusion proteins including PTD and LIM degradation proteins are provided as one embodiment of the present invention. Fusion proteins can include PTD and one or more LIM degradation proteins or polypeptides. Useful LIM degradation proteins include LMPs disclosed in US Pat. Nos. 6,300,127, 6,444,803 and 6,521,750, and US Patent Application 09 / 959,578 (filed April 28, 2000). Suitably LMP is RLMP, HLMP-1, HLMP-1s, HLMP-2, HLMP-3 or a peptide derived therefrom. Such peptides include polypeptides such as 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 nucleotide chain encoding the LIM degradation protein is suitably mixed with the nucleic acid molecule at standard state to complete the following chain lengths:

tcctcatccg ggtcttgcat gaactcggtg:

Or mixed with nucleic acid rich in a highly regulated state to complete the following chains:

gcccccgccc gctgacagcg ccccgcaa,

Or both.

The "standard mixed state" depends on the size of the probe, the environment and concentration of the nucleic acid reactant, and the mixed state (in the original state, the right blot, the mixing of the DNA-RNA hybrid: the prime blot). Determination of the "standard mixed state" is well known to those skilled in the art. This method is described in US 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).

Premix the blot at 42 ° C. for 2 hours in 50% formamide with one set of standard mix, 5 × SSPE (150 nM NaCl, 10 mM Na H ₂ PO ₄ [pH 7.4], 1 mM EDTA [pH 8.0]). ]), 10% dextran sulfate, 1% SDS and 100 micrograms / ml Salmon Sump DNA. A32P-cDNA probe are added and mixing continued for 14 hours. Then wash twice with 2X SSPE, 0.1% SDS at 22 ° C. for 20 minutes and then with 0.1 × SSPE, 0.1% SDS at 65 ° C. for 1 hour.

In the "very regulated state", when the two chains are nearly identical, the probe will mix the target chains. Such techniques in which nearly identical chains are mixed but non-identical chains are not known are already known in the art.

"Protein" is considered to include analogues thereof, and "amino acid" is intended to include L-form, D-form, and modified amino acids. These substitutions are well known to those skilled in the art and use structural intermolecular analogs. Amino acid sequences also include peptide or protein sequences comprising N-terminal or C-terminal amino acids in the indicated sequences. A "bone guide protein" is one that includes a variant or biologically active fragment of a polypeptide and a protein of full length.

It is well known to those skilled in the art that a single amino acid is encoded by one or more nucleotide codons and that the nucleotide sequence is modified to produce an alternative nucleotide sequence that encodes the same peptide. Thus, a modified embodiment of the present invention includes an alternative DNA sequence encoding a peptide containing the above amino acid sequence. DNA sequences encoding peptides containing the desired amino acid sequence include DNA sequences encoding the combination of the desired sequence located at the N- or C-terminus with other amino acids to the desired amino acid sequence. The amino acid and nucleic acid sequences may comprise additional residues, in particular N- or C-terminal amino acids or 5 'or 3' nucleotide sequences, which sequences are more particularly when conferring osteoinduction for the expressed polypeptide or protein. It is important.

Additional nucleic acid bases may be added to the 5 'or 3' of the nucleotide sequence encoding the osteoinductive polypeptide, and may contain multiple DNA sequences such as promoters, polyadenylation signals, additional restriction enzyme sites, polycloning sites, other coding fragments, and the like. It can also be combined with. Thus, the overall length of these polynucleotides can vary greatly.

The "variant" of a polypeptide is not exactly the same as the original protein. For example, a variant of an osteoinductive polypeptide or protein is obtained by modifying an amino acid sequence by inserting, deleting or replacing one or more amino acids. The amino acid sequence of a polypeptide or protein can be modified by substitution to produce a polypeptide having almost the same or improved quality compared to the original polypeptide. Substitutions may be conservative substitutions. "Preservation substitution" refers to the substitution of an amino acid with another amino acid having a side chain of similar polar / nonpolar, charge or size. The twenty important amino acids have nonpolar side chains (alanine, valine, leucine, isoleucine, proline, phenylalanine, and tryptophan), those with non-charged polar side chains (methionine, glycine, serine, threonine, cystine, tyrosine, asparagine, And glutamine), those with acidic side chains (aspartate, glutamate) and those with basic side chains (lysine, arginine, and histidine). Conservative substituents are described, for example, in Glu, Asp for Asn; His for Lys, Arg or Phe; Asn for Gln, Asp or Glu; And Ser for Cys, Vy or Gly.

One skilled in the art can obtain a modified polypeptide by substituting the first amino acid for a second amino acid at one or more positions of the polypeptide structure to have biological activity. Amino acid substitutions can increase biological activity by causing structural transformations in polypeptides.

Experts can substitute amino acid sequences based on amino acid hydrophilicity index or hydropathic index. Thus, modified amino acid molecules of the present invention are 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 At least 100% and at least about 80-90% amino acid sequence is identical to the amino acid comprising the amino acid sequence of the polypeptide of SEQ ID NO 7, or SEQ ID NO 8, suitably at least about 80-90%. Thus, the amino acid sequence of the modified osteoinductive polypeptide or protein is nearly identical to the original osteoinductive polypeptide or protein amino acid sequence. "Almost identical" means 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 sequence that induces biological and enzymatic activity similar to that produced by an osteoinductive polypeptide or protein comprising a polypeptide in SEQ ID NO 8, which activity is at least 70 of the original osteoinductive protein %, More suitably 100%.

Variants of the osteoinductive protein include 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 Amino acid residues that are not present in the corresponding osteoinductive protein comprising NO 7, or SEQ ID NO 8 may comprise LMP-1, LMP-2, LMP-3, SEQ ID NO 1, SEQ ID NO 2, SEQ It may also be deleted in comparison to an osteoinductive protein comprising ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, or SEQ ID NO 8. Variants also include 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 truncated "fragment" as compared to the corresponding protein comprising SEQ ID NO 8. A fragment is only part of a full length protein or polypeptide.

Bone morphogenic proteins (BMPs) are members of the TGF-beta superfamily of proteins. BMPs have been shown to cause osseous or cartilage formation. In accordance with the present invention, fusion proteins and bone morphogenic proteins of PTD are provided. BMPs are 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 morphogenic proteins BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9 are particularly useful in the present invention.

Smad proteins are intercellular proteins that mediate signals from receptors for extracellular TGF-beta-related factors (Heldin, et al., "TGF-beta Signals from Cell Membrane to Nucleus Via SMAD Proteins," Nature, Vol. .390 (1997). Smad proteins can be activated by binding BMP to its receptor (eg, phosphorylation). When activated, Smad proteins translocate to the nucleus and control gene expression. PTD fusion proteins and Smad proteins are also provided herein. Smad-1, Smad-2, Smad-3, Smad-4, Smad-5, Smad-6, Smad-7, Smad-8 are particularly useful for enhancing bone induction, which contain a protein transduction domain in the present invention. One fusion protein is transferred.

The protein transduction domain according to the present invention may be any peptide, analog, or peptide nucleic acid (PNA) sequence capable of transferring intracellularly attached proteins, peptides or nucleic acids across the plasma membrane of the cell. It has been found by the inventors that osteoinductive proteins can be migrated into cells without damaging them to enhance osteoinduction and proteoglycan synthesis. PTDs include, for example, polypeptides derived from the Drosophila homeostatic transcription factor Antenapedia (Antp), herpes simplex virus (HSV) protein VP22, signal peptide sequences such as Kaposi's fibroblast growth factor (K-FGF) (Lin). , et al., J. Biol. Chem., Vol. 270, p. 14255-14258, 1995), membrane displacement sequences derived from K-FGF signal peptide sequences (Rojas, et al., Nat. Biotech., Vol. .16, p.370-375, 1998), and human immunodeficiency virus (HIV) -1 transcription activator TAT (Fawell, et al., Proc. Natl. Acad. Sci. USA, Vol. 91, p.664 -668, 1994). PTD is described in US Pat. No. 5,652,122 and Schwarze et al., “Protein Conversion: Non-Limited Migration to All Cells,” Trends in Cell Biology, Vol. 10 (2000). The inventors have found that HIV-TAT PTD is particularly useful for the present invention.

The nucleic acid comprises a nucleotide sequence that encodes a fusion protein functionally linked to a promoter, the fusion protein comprising a protein transduction domain (PTD) and an osteoinductive protein. The nucleic acid can be part of a vector (eg, an expression vector such as a plasmid). Osteoinduced proteins can include LIM degradation proteins, bone morphogenic 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 .

Also provided by the present invention are methods for delivering osteoinduced proteins to cells. In the method of the present invention, at least one osteoinductive protein can be delivered to the cell via transformation, wherein the fusion protein comprises a protein transduction domain (PTD) and an osteoinductive protein and is in contact with the cell such that the fusion protein is in the cell Is transferred to. Transfer is promoted by protein transduction domains or cell permeable peptides.

Cells into which the osteoinductive protein can be transferred include bone (ie, bone formation) and non-osteoblast cells. Such cells include, for example, buffy coat cells, stem cells (e.g., mesenchymal stem cells, multipotent and pluripotent cells), intervertebral disc cells (e.g., round fibro cells and nuclear pulposus), mesenchymal cells , Hematopoietic cells, endothelial cells, and muscle cells. Stem cells can be derived from autologous or allograft tissue.

Also provided are cells that are converted or expressed by a fusion protein of a protein transduction domain (PTD) and a osteoinductive protein. Such cells include buffy coat cells, stem cells (e.g., mesenchymal stem cells, pluripotent and pluripotent cells), intervertebral disc cells (e.g., round fibro cells and nuclear pulposus), mesenchymal cells, Hematopoietic cells, endothelial cells, and muscle cells. Cells containing the fusion proteins of PTD and osteoinduction proteins as described above can be transplanted into the body of a mammal to induce bone formation. Methods for causing bone formation using LMPs as osteoinductive proteins are described in US Pat. No. 6,300,127. Cells comprising fusion proteins of PTD and osteoinductive proteins can also be implanted in intervertebral discs to stimulate proteoglycan and / or collagen synthesis, as described in US Patent Application No. 10 / 292,951 on November 13, 2002.

Also provided are conjugates of PTDs and nucleic acids comprising nucleotide sequences encoding osteoinductive proteins. PTD / nucleic acid conjugates can be used for direct overexpression of osteoinductive proteins to enhance bone formation or intervertebral disc regeneration. Osteoinduced proteins encoded by nucleic acid sequences include LMPs, BMPs, Smad, and the like. Methods of chemically binding peptides to nucleic acids are well known. One such method is disclosed in US Pat. No. 5,652,122. The nucleic acid may be in the form of an expression vector comprising a nucleotide sequence that encodes an osteoinductive protein linked to a promoter.

The method of the present invention can be used to induce expression of one or more bone morphogenic proteins or modified growth factor beta proteins in cells as described in US Pat. Appl. No. 10 / 382,844, issued March 7, 2003. Expression of one or more proteins selected from the group consisting of BMP-2, BMP-4, BMP-6, BMP-7, TGF-β1 and combinations thereof may be used to express the cells with a fusion protein comprising PTD and osteoinductive protein according to the present invention. By contact. In addition, according to the present invention, cells which overexpress one or more proteins selected from the group consisting of BMP-2, BMP-4, BMP-6, BMP-7, TGF-β1 and combinations thereof may be provided. Such cells can be any somatic cell, including stem cells, buffy coat cells, bone marrow transplant cells, peripheral blood cells or adipocytes. The cells can also be stem cells derived from autologous or allograft tissue.

In the kidney, cells can be stimulated through administration of a PTD / LMP fusion polypeptide according to the invention to produce BMP-7, and stem cells can be treated as PTD / LMP fusion polypeptides to induce BMP-7. Once implanted into the kidney, the production of BMP-7 may provide a treatment that would be impossible with exogenes BMP-7 administration. BMP-7 can repair severely damaged tubular epitellial cells (Zeisberg, et al., Nat. Med. 9: 964-968, 2000). It has been reported that one cause of chronic kidney disease, asthma, and bone disorders can be successfully treated by administering BMP-7 to mice (Lund, et al., J Am Soc Nephrol 15 (2): 359-369, 2004). . In a rat genetic model of chronic kidney disease and fibrosis, renal dysfunction was restored by administering exogenes human recombinant BMP-7 (Zeisberg, et al., Am J Physiol Renal Physiol 285 (6): F1060-7, 2003). BMP-7 is also known to partially restore renal hypertrophy due to diabetes (Wang, et al., Kidney Int 63 (6): 2037-2049, 2003). Davis and colleagues have shown that BMP-7 deficiency is a pathophysiological factor in chronic kidney disease (J Am Soc Nephrol 14 (6): 1559-1567, 2003). LMP-1 induces the production of BMP-7 by this study and the inventors, and LMP peptide (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) function similar to the LMP-1 protein, and include a fusion polypeptide comprising a protein transduction domain and an LMP-1 or LMP-3 protein, or related peptides. It can be seen that it is useful for the treatment of kidney disease. Stem cells induced to express BMP-7 by the fusion polypeptide according to the invention are partially useful for this purpose.

BMP-4 and BMP-7 have also been found to inhibit proliferation and induce apoptosis in human myeloma cells. Therapies for BMP have been proposed for myeloma bone disease and myeloma cell proliferation. The method of the present invention comprises LMP-1, LMP-3, or 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 in vivo. , And a method for stimulating BMP-4 and BMP-7 by administering by injection of a peptide comprising SEQ ID NO 8 or by transplantation or transplantation of stem cells treated as a PTD / LMP fusion polypeptide. Combinations of implantable drug delivery devices with stem cell implants incorporating the source of PTD / LMP may be partially effective.

Stem cells or multipotent progenitor cells can provide a cell source capable of producing osteoblasts. These cells are separated by several stages of differentiation and induce differentiation in certain lineage pathways. Such cells can be used for the treatment of bone diseases such as osteoporosis or osteogenesis incomplete and non-therapeutic fractures. Core binding factor alpha1 (Cbfa1) was found to be required for osteogenics. BMP-2, BMP-4 and BMP-7, known to cause osteoblast differentiation, further regulate Cbfa1 expression. BMP-8 and Smad-3 are more regulated during osteoblast differentiation. The activity of TGF-beta / BMP-Smad-3 signaling has been shown to promote Cbfa1 expression and osteoblast differentiation. The present invention provides functional BMPs, LMPs, Smad proteins or combinations thereof to enhance osteoblast differentiation in cells, such as, for example, mesodermal progenitor cells due to human bone marrow transplantation. Suitable cells include the multipotent cells disclosed by Jiang et al. (Nature, Vol. 418, p. 41-49). Administration of suitable osteoinducing proteins or polypeptides or combinations thereof may be ex vivo prior to cell transplantation, or may be in vivo via transplantation or injection.

For in vivo administration, the bone-induced proteins of the present invention are injected into the target site and transported internally near the cell via PTD or cell permeable peptide. Alternatively, carriers may be used in combination with PTD / osinduced polypeptide. The implant may comprise a reservoir for locating the PTD / induced polypeptide to release to surrounding tissue, or may comprise a porous composition that is contained in a solution comprising one or more PTD / induced polypeptide constructs. Hydrogels, time-release capsules or spheres, liposomes, microspheres, nanospheres, biodegradable polymers or other drug delivery systems can be applied to transfer peptides and proteins of the invention to target cells and tissues. For example, US Pat. No. 6,475,516 (DiCosmo, et al.) Provides a hydrogel containing a liposome therapeutic agent, such as an antibiotic, wherein the hydrogel is shared with the surface of a dripping medical device, such as an implant. Are combined.

Characteristic of intervertebral disc degeneration is the reduction of proteoglycans in intervertebral discs, in particular the reduction of sulfide glycosamine oglycans (sGAG) and aggrecans. The rate of aggrecan production and a decrease in the major proteoglycans of the intervertebral discs are the main cause of disc degeneration. Because of the important role of proteoglycans in the intervertebral discs, recovery of normal proteoglycan production in the intervertebral discs is very important in all biological treatments of intervertebral disc degeneration.

The inventors have found that, by experiments, intervertebral disc proteoglycan production is increased in vivo and in vitro upon LMP-1 overexpression or intercellular administration. LMP-1 overexpression increases the expression of BMP-2 and BMP-7 mRNA in vivo and in vitro. Nogin, which inhibits BMP-2 and BMP-7, inhibits the expression of proteoglycans by AdLMP-1, which means that LMP-1 induced by the expression of proteoglycans is delivered by the expression of BMPs. LMP-1 administration by gene therapy or protein therapy can thus be used to stimulate proteoglycan production in the intervertebral disc to serve as a therapeutic action of intervertebral disc regeneration.

Cytokines, such as TGF-β1, IGF-1, and EGF, have been known to stimulate intervertebral cell mitosis, causing some production of proteoglycans. Cytokines such as BMP-2 and BMP-7 are also known to stimulate the production of proteoglycans. However, because cytokines are small water-soluble molecules, they rapidly diffuse out of the intervertebral discs or are inactivated by other regulatory factors. LIM degradation protein-1 (LMP-1) is an intercellular regulatory molecule known to secrete multiple, multiple BMPs from leucosite and osteoblasts. By transporting LMP-1, LMP-2, LMP-3 or osteoinductive peptides from LMP-1 or LMP-3, or a combination thereof into cells, BMP production is stimulated intracellularly, in particular via PTD / nucleic acid conjugates Can be. Suitable osteoinductive peptides include polypeptides from 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 Can be mentioned.

The invention will be more readily understood through the following examples.

FIG. 1 is a graph showing the amount of BMP protein in the medium after 6 days of treatment of ring cells with LMP-1 (AdLMP-1 at MOI 25). BMP-2 and BMP-7 increased significantly but BMP-4 and BMP-6 did not differ significantly from the media of untreated control cells. Each result is shown as a ratio of amount to untreated cells. Mean values and SEM for 7 samples are shown (* p <0.05, ** p <0.01 compared to NT).

Figure 2 is a graph showing the amount of BMP mRNA over time by treating the ring cells with LMP-1 (AdLMP-1 in MOI 25). The amount of BMP-2 mRNA is very irregular in the initial 12 hours after AdLMP-1 treatment and parallel after 3 days. The amount of BMP-7 mRNA increased significantly after 3 days of AdLMP-1 treatment. Each result is expressed as the ratio of the amount to untreated cells at the same time period. Mean values and SEM for six samples are shown (** p <0.01 compared to NT for response time).

3 is a graph of real-time PCR of the amount of aggrecan mRNA in ring cells treated with LMP-1 (AdLMP-1). Agrecan mRNA levels increased significantly after 6 days of treatment with AdLMP-1 at MOI 25 compared to untreated cells. The amount of mRNA of egrecan was not changed in cells treated with AdLacZ compared to untreated cells. Each result is expressed as a positive ratio compared to untreated cells and expressed as mean and SEM for 9 samples. AdLMP-1: MOI 25, AdLacZ: MOI 25 (** p <0.01 compared to NT).

FIG. 4 is a table summarizing the parameters and results of administration of LMP-1 via PTD / LMP-1 fusion proteins in Harlan amic mice.

FIG. 5 is a table summarizing the parameters and results of administration of LMP-1 through PTD / LMP-1 fusion proteins in New Zealand white rabbits.

FIG. 6 is a table summarizing the results of administration of osteoinductive peptides via PTD / LMP-1 fusion protein to Harlan's amic mice and New Zealand white rabbits, clearly showing that bone growth is detected by X-rays.

Synthesis and use of (His) 6 TAT-LMP comprising the protein transduction domain of HIV-Tat and the LMP-1 protein is as follows. The pTAT-HA-vector is obtained from the University of Washington (St. Louis).

The NcoI restriction site is added to the 5 'end of hLMP-1 using pCDNA3.1 / hLMP-1 as a template for PCR with the following primers:

Fwd: 5'-CCATGGAfICCfICAAAGTAGTGC-3 '

Rev: 5'-CAGGGCGGGCGGCTGGTAG-3 '

The reaction was carried out at 95 ° C. for 2 minutes [95 ° C., 30 seconds; 66 ° C., 30 seconds; 10 mm at 72 ° C, 1 mm] * 25, and 72 ° C. The PCR product is cloned into a PCRII-TOPO vector (Invitrogen) to identify appropriate copies as sequences.

The structure of the (His) ₆ TAT-LMP vector is completed by restriction endonuclease digestion of plasmid clones including NcoI and ClaI, and the final product is purified by agarose gel electrophoresis and electrolysis. Full length hLMP-1 sequences are isolated by restriction digestion of pCDNA3.1 / hLMP-1 vectors, including ClaI and EcoRI, and purified by agarose gel electrophoresis and electrolysis. The pTAT-HA-vector is also digested as NcoI and EcoRI and the final linear vector is purified by agarose gel electrophoresis and electrolysis. The product is then regated by standard method overnight at 16 ° C. Properly regated product (5'hLMP-1 + 3'hLMP-1 + linear pTAT-HA-vector = (His) 6 TAT-LMP vector) is determined by sequence agarose gel electrophoresis and molecular weight determination.

Ligation products are infected with BL21 (DE3) satisfying cells to identify appropriate clones by restriction assays.

Synthesis and Cultivation of (His) ₆ TAT-LMP Protein

Appropriate BL21 (DE3) Escherichia coli containing active clones of the (His) ₆ TAT-LMP fusion construct was grown to 0.8 OD (600 nm) and induced protein production for 4 hours at 37 ° C. with 100 μM IPTG. do. Induced cells are grown by standard methods and lysed by sonication (4 * 20 s, each with 2 mm residual, 4 ° C.).

And the lysate centrifuged (10000 * g) Ni ^{2 +} Sepharose affinity column (Invitrogen) and the resulting supernatant was purified in a gravity flow condition by. The column is washed (20 mM PO4 buffer, pH 6.0, 8 M Urea, 250 mM NaCl, 20 mM imidazole) and dissolved by separating the bound protein (20 mM PO4 buffer, pH 4.0, 8 M Urea, 500 mM NaCl).

Isolate the linear particles from 0% buffer B to 100% buffer B (40 mm @ 5 mL / min) (buffer A: 20 mM sodium carbide, pH 11, 8 M Urea; buffer B: 20 mM sodium carbide, pH 11, 8 M Urea, 2 M NaCl) is used for anion exchange chromatography (Hitrap Q HP, 5 mL, Pharmacia). The separated sections (5 mL) are analyzed for the amount of hLMP-1 by SDS-PAGE and Western blot analysis.

Sections active in hLMP-1 are subjected to hydrophobic attraction chromatography (Hitrap, Phenyl-sepharose, 5 mL, Pharmacia). The illumination (buffer A: 20 mM sodium carbide, pH 10.5, 1.5 M ammonium sulfide; buffer B: 20 mM sodium carbide, pH 10.5) is 20 mm @ 5 mL / mm with linear particles consisting of 0% to 100% buffer B. Is performed. The separated sections (5 mL) are analyzed for the amount of hLMP-1 by SDS-PAGE and Western blot analysis. Freeze-drying under reduced pressure until (His) ₆ TAT-LMP fusion protein is used.

Use of (His) _-6 TAT-LMP Fusion Protein for De novo Bone Formation

Lyophilized sections (55 pg) are resuspended in 40 mM KOH to make 1.0 μM solution. Human buffy coat cells are prepared (Viggeswarapu, et al., J. Bone Joint Surg., Vol. 83 (3), p. 364, 2001). The cells are mixed with 5 μL of (His) ₆ TAT-LMP fusion protein in alpha MEM (Gibco) and aged at 37 ° C. for 30 minutes.

Appropriate amount of human buffy coat cells containing (His) ₆ TAT-LMP fusion protein are added to the porous collagen matrix for transplantation. To demonstrate de novo bone formation in vivo, 100 μL of cell suspension is added to steril 5 * 5 mm type I human collagen via a steril pipette and transplanted into mice. An equal amount of cell suspension is added to 10 * 25 mm sheets to implant to promote vertebral fusion. The thoracic disc is surgically implanted subcutaneously into the chest of about 4-5 weeks of acemic rats (rnu- / rnu-). After 4 weeks the rats were killed and the intervertebral discs were removed and mixed with 70% ethanol, then analyzed by radiograph and analyzed by undichlide histolysis experiments (segmented to 5 μm and stained with Goldner Trichrome).

In rabbits, sterral lumbar arthroplasty is performed and a carrier matrix is implanted on each side of the spine containing 4 * 10 ⁶ transformed buffy coat cells (e.g. 15% hydroxyapatite / 85% Collagen sponge with calcium triphosphate). After 4 weeks, the rabbits are euthanized and their lumbar incisions are made. The spinal fusion is accessed by detection manual motion (indicating failed fusion), radiographs, CT scans, and any manual armations of the moving segments for decalidized histology.

In rats and rabbits, the radiographs show high levels of degraded bone formation for the morphology of the original collagen discs or for LMP-1 infected human buffy coat cells. In the controlled state no dissociative bone formation appeared and the original collagen disc or sheet was shown to be absorbed. The histology showed a new bone streak in line with the osteoblast in the LMP-1 transducing implant, whereas no bone was observed and the carrier was partially reabsorbed.

Use of osteoinductive peptides to induce bone formation in vivo

The protocol is the same as above. Peptides are applied to four separate implants (three for rabbits), with 250 microliters per disc, 6 M / ml. Implants include collagen discs. Implants are implanted in Harlan amic rats or New Zealand white rabbits. Dosages are 5 nM, 10 nM, 12.5 nM, 15 nM, 17.5 nM, 20 nM, 22.5 nM and 25 nM. The results are shown in FIG.

LMP-1 Stimulation of sGAG Synthesis

Lumbar intervertebral disc cells from Sprague Duli rats are treated and tested in vitro by treating the cells at various doses (0, 5, 10, 25, 50 infection concentrations) as LMP-1 nucleotide sequence inserts (AdLMP-1). LMP-1 overexpression effect was measured by incubation for 6 days in a monolayer in the tube. Glycosamine oglycans in the medium are quantitatively determined using the DMMB method. Real-time PCR is used to quantitate mRNA levels of agrecan, overexpressing LMP-1, BMP-2, 4, 6 and 7. Quantify the amounts of BMP-2, 4, 6 and 7 in the medium using the direct ELISA method. To demonstrate that LMP-1 expression of the disc cell proteoglycan product includes BMPs, noggins that specifically block BMP-2,4,6 and 7 activity were added to AdLMP-1 treated cells at various concentrations. do.

In vivo gene therapy experiments are performed in New Zealand white rabbits. Adenoviruses containing the AdLMP-1 (experimental) or marker gene (AdLacZ-control) are injected into the lumbar disc at three doses (10 ⁶ , 10 ⁷ and 10 ⁸ pfu / disc). Three weeks after incubation of the injected disks, the mRNA levels of total LMP-1 (endogenous), overexpressing LMP-1, Agrecan, BMP-2 and BMP-7 are measured.

AdLMP-1 in various infections (MOI) of 25 is sufficient to induce maximum of sGAG expression. Six days in monolayer culture are required to obtain the maximum amount of sGAG after AdLMP-1 treatment (MOI 25). Aggrecan mRNA is up to twofold compared to the control. When cells were cultured in alginic acid, the effect of AdLMP-1 treatment on sGAG product was maintained for 3 days. After 6 days of AdLMP-1 treatment, the amount of BMP-2 and BMP-7 mRNA increased significantly by 3.0 ± 0.2 and 2.8 ± 0.3 with respect to the control (p <0.01). The amount of BMP-4 and BMP-6 mRNA does not change. The amount of BMP-2 and BMP-7 proteins in the medium is significantly increased compared to the control (p <0.01). In comparison, the amount of BMP-4 and BMP-6 proteins does not increase. Noggin completely blocks the expression of proteoglycans by AdLMP-1 at 3200 ng / ml. Endogenus amount of LMP-1 mRNA is measured in lumbar nuclear pulposus. Discs injected with 107 pfu / disc of AdLMP-1 in vivo have a significant increase in the amount of LMP-1, BMP-2 and BMP-7 mRNA compared to the control.

In vitro experiments are performed on Sprague Dooly rat lumbar disc cells. Two labeled t-tests are used to compare the experimental groups to the controls. Error bars in the numerical values represent 1 SEM.

Alternative defective type 5 adenoviruses carrying cDNA for the human LMP-1 gene are used (AdLMP-1). Monolayer culture experiments of SD ring cells are performed to determine the relationship between virus dose and overexpression LMP-1 mRNA by using real-time PCR with primers specific for virus transfer LMP-1 cDNA. Monolayer culture experiments are performed on SD ring cells to determine the relationship between viral dose and sGAG product. The amount of sGAG is measured using the DMMB method. The optimal dose of AdLMP-1 is defined as the lowest dose reaching the mean of sGAG production. Real-time experiments (9 days) are performed using the optimal dose (25 MOI) to determine the minimum time (6 days) required to reach a flat level of sGAG production. The optimal virus dose and the time determination (AdLMP-1 at 25 MOI and 6 days post treatment) are used to compare the amount of agrecan mRNA in AdLMP-1 treated cells with the control. To determine if nuclear cells responded to circulating cells in a similar form, the AdLMP-1 effect and cell number in sGAG production were determined for cyclic and nuclear cells at 25 MOI 6 days after treatment. To determine the long-term effects of AdLMP-1 treatment, sGAG accumulated in alginic acid of SD ring cells cultured in alginic acid is measured every 1, 2, and 3 weeks.

Monolayer culture experiments on SD ring cells are performed using AdLMP-1 at MOI 25. MRNA levels of LMP-1, BMP-2, BMP-4, BMP-6 and BMP-7 are measured by real-time PCR assays every 0.5, 1, 3 and 6 days with AdLMP-1 treatment. The amounts of BMP-2, BMP-4, BMP-6 and BMP-7 are measured in the medium 6 days after treatment using the direct ELISA method. Noggin is added to the medium at the beginning of AdLMP-1 treatment to determine the blocking effect of BMP activity. Changes in sGAG content at various concentrations after treatment with AdLMP-1 are measured on day 6 of the experiment.

Four New Zealand white rabbits are used to determine the effect of LMP-1 (via AdLMP-1) in vivo administration on Agrecan, BMP-2 and BMP-7. Front lumbar discs L2 / 3, L3 / 4, L4 / 5 and L5 / 6 are exposed with the left rotifertoniyl approach. Experimental virus (AdLMP-1) or control virus (AdGFP-type 5 adenovirus with green fluorescent protein as insert) is injected into each exposed disk nucleus in different forms in 10 ⁷ plaque forming units (pfu) (eg , AdLMP-1 on the two disks, AdGFP on the other two disks). The virus is administered as 10 microliters of phosphate buffered saline via 30G Hamilton syringe. Three weeks later, the nuclear pulposus tissue from the injected lumbar disc is incubated. Nuclear tissues from both rabbits are congested into a control or experimental disc group to obtain sufficient mRNA for further analysis. Reverse transcription and real-time PCR are used to quantify the mRNA levels of total LMP-1, BMP-7 and Aggrecan. Primers for total LMP-1 are designed to identify endogeneus and overexpression LMP-1.

In a second in vivo experiment, several doses of AdLMP-1 virus are used to determine the dose response relationship. AdLMP-1 at three doses (10 ⁶ , 10 ⁷ , 10 ⁸ pfu) and AdGFP at a single dose (10 ⁷ pfu) are administered. In this experiment, all disks for each animal are injected with a single dose of virus instead of the replacement virus as in the experiment. Eight rabbits using four groups of viruses each for two rabbits are used. The rabbits are euthanized after 3 weeks and the nuclear Fulposus is grown. Each treatment group congested the cultured tissue from and allowed mRNA to be isolated to produce the corresponding cDNA. Real-time PCR is used to quantitate mRNA levels of total LMP-1, overexpressing LMP-1, BMP-2, BMP-7 and agrecan.

After 12 hours of treatment with the virus at different doses (MOI 0, 10, 25, and 50), the relative amount of overexpressed LMP-1 mRNA was leveled to achieve the lowest amount of overexpressed LMP-1 mRNA (AdLMP-1). To indicate. Increasing the dose of AdLMP-1 significantly increases the amount of overexpressed LMP-1 mRNA compared to MOI 5. There was no overexpression LMP-1 mRNA that could be found in the untreated or AdLacZ controls, indicating that AdLMP-1 caused the overexpression of LMP-1 in the circulating cells depending on the dose.

SGSG concentration in medium taken from round cells is measured after 6 days of treatment with AdLMP-1 according to different doses. Day 6 sGAG concentration is a measure of the amount of total sGAG induced by cells over the three days between media modification and sGAG measurements. Data is presented as percentages for the treated and untreated controls. Administration of LMP-1 (AdLMP-1 at MOI 25) provides a maximum sGAG amount of 3.1 ± 0.2 fold over the control (p <0.01). There is no significant change in the amount of sGAG in the medium between cells treated with MOI 25 or MOI 50. The AdLMP-1 dose of MOI 25 is the lowest value for which the maximum sGAG response can be obtained, so select MOI 25 as the working dose for the remaining experiments.

Time-lapse experiments are performed to determine the effect of different experiment lengths after treatment at MOI 25 with AdLMP-1. Ring cells are treated with LMP-1 (AdLMP-1 at MOI 25) and production of sGAG is measured over 3 days. Results are normalized to the amount of DNA at each point and expressed as a ratio with untreated control at the same time. The amount of sGAG increased to 1.6 ± 0.2 (p <0.01) on day 3, 2.9 ± 0.1 (p <0.01) on day 6 and 2.8 ± 0.1 (p <0.01) on day 9. The sGAG does not change between days 6 and 9, indicating that planarization occurs on day 6.

LMP-1 Induction of Aggrecan Synthesis

Since agrecan is the main proteoglycan of the intervertebral disc, the amount of agrecan core protein mRNA is measured 6 days after treatment with AdLMP-1. Quantitative Real-time PCR is used and data is presented as a percentage of untreated controls. After AdLMP-1 (MOI 25) treatment, the agrecan mRNA amount increased 2.1 ± 0.1 (p <0.01) relative to the control. After AdLacZ treatment (MOI 25), the amount of agrecan mRNA remained unchanged and showed an increase of 1.0 ± 0.16 for the control group. These results, together with sGAG experiments, indicate that LMP-1 stimulates intervertebral disc cell production of proteoglycans.

By determining the dose and time for optimal AdLMP-1 activity in the culture system, the effect of LMP-1 was compared by administering AdLMP-1 to neutrophil cells. Rat round and nuclear cells were treated with AdLMP-1 at MOI 25 to determine the sGAG concentration for each cell at day 6. sGAG concentrations are normalized to the amount of DNA to calculate the lowest deviation in cell number. Results are denormalized to untreated controls and compared to controls. The results for untreated cells showed 0.6 ± 0.04 sGAG / DNA and 1.0 ± 0.12 sGAG / DNA, respectively. This result shows a large increase for each untreated control. Relative values with untreated controls for AdLMP-1 treated cells are similar between cyclic and nuclear cells. As a result of measuring DNA at 6 days after treatment, the amount of ring cell DNA increased 1.2 fold over the untreated control group (p <0.01), but the nuclear cells were not infected with AdLMP-1. This indicates that LMP-1 produces a small but significant increase for ring cells.

To test the effect of administration of LMP-1 in vitro over several weeks, an alginic acid culture system was used. Alginic acid provides a three-dimensional matrix of cells important for long-term maintenance of chondrogenic phenotypes in in vitro culture. Ring cells grown in monolayer are treated with AdLMP-1 at MOI 25 and transferred to alginic acid medium after 24 hours. Cells are cultured for 1, 2 and 3 weeks. At week 1 the sGAG amount in AdLMP-1 was 1.5 ± 0.06 (p <0.01) compared to the untreated control (FIG. 5) and at week 2 2.9 ± 0.3 (p <0.01). This difference persists at week 3 and shows 2.9 ± 0.1 (p <0.01). This means that the effect of AdLMP-1 continues to increase for at least 3 weeks in alginic acid.

By showing the effect of LMP-1 overexpression in proteoglycans, the mechanism of LMP-1 overexpression was revealed. The change over time in the mRNA of overexpressing LMP-1 and BMPs (BMP-2, 4, 6 and 7) is measured. It can be seen that BMPs are stimulated in leucosite and osteoblast by LMP-1 overexpression.

The change in overexpression LMP-1 over time after the treated cells were treated with MOL 25 with AdLMP-1 was measured. Since the data do not show any overexpressed LMP-1 mRNA in the control group, the data is expressed as the ratio of the maximum mRNA amount of overexpressed LMP-1 mRNA instead of the ratio to the untreated control group (day 6). LMP-1 mRNA is detected 12 hours after treatment and continues to increase until day 6. This indicates that LMP-1 takes effect at the bottom of the gene within 12 hours of administration of LMP-1.

The amount of BMP mRNA after treatment with AdLMP-1 at MOI 25 was measured by real-time PCR over time and expressed as a ratio with the control for each time point. BMP-2 mRNA is expressed early and increases significantly after 12 hours of treatment with LMP-1. BMP-2 mRNA is flattened on day 3. BMP-7 mRNA is expressed after BMP-2 and increases until day 3 and day 6. BMP-4 and BMp-6 mRNAs do not change in comparison to untreated controls.

Based on the fact that LMP-1 mRNA overexpression causes the expression of BMP-2 and BMP-7 mRNA in circulating cells, it is tested whether these mRNAs increase with increasing BMP protein secretion in the medium. Quantify the amounts of BMp-2,4,6, and 7 in the medium by ELISA assay. Monolayer cultured round cells are treated with AdLMP-1 at MOI 25. The medium is changed every 3 days and analyzed on day 6. Samples containing BMPs secreted from the medium for 3 days are tested. The protein amounts of BMP-2 and BMP-7 showed an increase of 3.5 ± 0.4 (p <0.01) and 2.5 ± 0.3 (p <0.01), respectively, compared to untreated controls. BMp-4 and BMP-6 were unchanged.

Since the mRNA and proteins of BMP-2 and BMp-7 are expressed by LMP-1, it is tested whether blocking these BMPs prevents the increase in sGAG induced by LMP-1. Ring cells treated with MOL 25 with AdLMP-1 were simultaneously treated with BMP inhibitor nogin in the medium to measure the amount of sGAG on day 6. Cells treated with AdLMP-1 alone increased the amount of sGAG by 2.7 ± 0.3, while cells treated with 3200 ng / ml of nogin and AdLMP-1 in MOI 25 increased by 1.1 ± 0.1, indicating that Nogin was AdLMP. -1 means to completely block the effect. Cells treated with Noginman showed an increase of 0.8 ± 0.1 with little change. The inhibitory effect of Noggin on AdLMP-1 leading to sGAG production is concentration dependent.

Endogenus amounts of aggrecan, BMP-7 and LMP-1 mRNA in control discs (AdGFP injected discs) are measured in nuclear pulposus. Endogenus mRNA levels are used to measure the increase in aggrecan, BMP-7 and LMP-1 mRNA. Discs injected with AdLMP-1 had a 830% increase in total LMP-1 mRNA levels compared to discs injected with AdGFP. Administration of LMP-1 by AdLMP-1 increased the amount of BMP-7 mRNA by 1100% compared to the overall control (p <0.05). Aggrecan mRNA amount was increased by 66% compared to the control.

Endogenus amounts of BMP-2, BMP-7, LMP-1 and Agrecan mRNA are also measured. Correlation between AdLMp-1 and LMP-1 mRNA is measured. By the administration of LMP-1 the amount of BMP-2 and BMP-7 mRNA is greatly increased up to a dose of 10 ⁷ pfu / disc. Aggrecan mRNA is increased by 50% by 10 ⁷ pfu / disc dose of AdLMP-1.

Administration of LMP-1 to intervertebral discs increases sGAG production and aggrecan mRNA. Administration of LMP-1 shows an increase in mRNA and protein of BMP-2 and BMP-7 in vitro. The effect of administration of LMP-1 on the expression of proteoglycans is inhibited by administration of BMP inhibitory noggin.

Sprague-Duli rats (11 months) are euthanized and the intervertebral disc cells are excised from the lumbar vertebra and cultured in sterilized state. Ring fibrosus and nuclear pulposus are separated and cut. Intervertebral disc tissue is placed in Dulbecco's Modified Eagle's medium containing 100 units / ml and 100 mg / ml streptomycin and Hams F12 medium (DMEM / F-12; GIBCO BRL, Grand Island, NY, USA). Tissues are treated at 37 ° C. in medium with 0.2% pronase (Sigma Chemical, St. Louis) for 1 hour followed by 0.025% collagenase (Sigma Chemical) at 6 hours 37 degrees. The isolated cells were washed and filtered through a 70 mm mesh (Falcon, USA) to filter 10% fat bobbin serum (FBS), 100 units / ml penicillin, 100 mg / ml streptomycin, 2 mM L-glutamine and 50 mg / ml ascorbate 75 cm ² with 12 ml DMEM / F-12 medium containing Put in a flask. Cells are grown at 37 ° C. in 5% carbon dioxide while maintaining humidity. Medium is changed every 2 days for 8 days.

Use two viruses. Experiments are performed using replication defective type 5 adenovirus comprising human LMP-1 cDNA induced by CMV promoter (AdLMP-1). The control virus consists of a replication defective type 5 adenovirus containing LacZ cDNA.

When the main medium of the intervertebral discs fuses, the cells are subcultured to 6-well plates at 400,000 cells per well. After 3 days, cells are treated with adenoviruses containing cDNA for human LMP-1 gene (AdLMP-1) or LacZ gene (AdLacZ). The number of initial cells is calculated using hemocytometa. Viral administration is indicated by the multipotency of infection. This is the number of recombinant adenovirus plaque forming units to which a single intervertebral disc cell is exposed. Cultured cells are treated at 37 ° C. with AdLMP-1 or AdLacZ of DMEM / F-12 with 0% FBS at various MOIs (0, 10, 25, 50). Culture doses are raised to 2.0 ml in DMEM / F-12 medium containing 1% FBS, 100 units / ml penicillin, 100 mg / ml streptomycin, 2 mM L-glutamine and 50 mg / ml vitamin C. Change the medium every 3 days.

The amount of sulfided glucosamine oglycans (sGAG) in the medium is measured using the 1,9-dimethylmethylene blue method (DMMB). Centrifuge 2 ml of medium (5000 * G, 30 min) to concentrate sGAG (using Centricon YM-50 centrifugal filter, Millefo Company, USA). Sample solution (20 ml) is mixed with DMMB die solution (200 ml) in a 96-well microtiter plate and the optimum density (OD) is immediately measured on a 520 nm wavelength filter. Standard curves are generated using a series of dilutions of chondroitin sulfate (Sigma Chemical). Total sGAG in the medium is normalized to the amount of DNA and expressed as a ratio with untreated control.

The number of cells is determined by the amount of DNA in each well and the amount of DNA is determined by the Chast Die 33258 (Polysciences, USA). Cultured cells are exposed to papain (10 units / ml). Pellet the cells and incubate at 60 ° C. for 3 hours. Twenty microliters alicut of papain digestion are mixed with a solution of the greast die 33258 in a 96-well fluoroplate. Emission and excitation spectra are measured as 456 nm and 365 nm in the fluorescence spectrometa LS 50B (Perkin-Elmer, USA). Standard curves are prepared using concentrations of calfmouth DNA (Sigma Chemical, USA).

Alginic acid bead medium is useful for maintaining long-term chondrogenic phenotypes. This method is used to determine the effect of AdLMP-1 on cultures for 3 weeks. Cells are released by trypsinization after one day and washed twice as medium. The isolated cells are resuspended in 0.6% low viscosity steryl alginic acid (Sigma Chemical) at 600,000 cells / ml. The cells are dispensed into 10 ml plastic syringes using a 21-gauge needle with 0.102 M CaCl + 0.15 M NaCl solution to form alginic acid beads. After 10 minutes, the newly formed beads (about 12,000 cells / beads) are washed three times with steril 0.9% sodium chloride solution and twice with DMEM / F-12. Beads containing round fibrosus cells were placed in 6 wells with DMEM / F-12 containing 1% FBS, 100 units / ml penicillin, 100 mg / ml streptomycin, 2 mM L-glutamine, and 50 mg / ml vitamin C. Incubate isolation from the plate. Medium is changed every two days over several periods (1, 2, and 3 weeks). Alginic acid beads are dissolved in 350 ml sodium citric acid buffer (55 mmol / L Na-citric acid, 50 mmol / L EDTA, 150 mmol / L NaCl, pH7.4). Cells are pelleted by centrifugation and the amount of sGAG in solution is measured using the DNMMB method. The amount of sGAG is negligible compared to the suspension. The results show that the sGAG of the solution was doubled compared to the untreated control.

Quantification of mRNA Volume

Real-time PCR is used to quantitatively measure mRNA levels of BMP-2, BMP-4, BMP-6, BMP-7 and overexpressing LMP-1. Primers for all genes are identified by amplicon sequence DNA by measuring product size in agarose gels. The 18S amount is measured in each sample and used as an internal control.

The total RNA of each sample is extracted by the consistent method using guanidium thiocinate-phenol-chloroform technology. The concentration of the isolated RNA is measured at 260 nm using spectrophotometer (DU-500; Beckman, USA). RNA is removed using DNAse1 (Ambion, USA) to remove DNA from the sample. Reverse transcription was treated with a 40 ml volume containing 2 mg total RNA, which included 30 U Avian Myeloblastosis virus reverse transcriptase (Promega, USA); 1 mM of oxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanidine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), and 1 mg oligo (dT) 15 primers are performed 30 cycles (95 ° C 30 "; 62 ° C 30"; 72 ° C 45 ") as Ampltaq DNA polymerase at 42 ° C for 45 minutes. RNA treated without reverse transcriptase to ensure DNA removal Samples are measured by PCR.

Real-time PCR is reported to be a fast and reliable reproducible method for quantitative determination of specific mRNAs. MRNAs of BMP-2, BMP-4, BMP-6, BMP-7 and Agrecan are quantitatively measured using the SYBR Green Real-Time PCR Kit (Applied Biosystem, USA). 25 ml of reaction including 5 ml of cDNA, 3.75 picomolar primers (BMP-2, 4, 6, 7 and 18S) and 12.5 ml of SYBR Green Master Mix (2x, Biorad, USA). To quantitatively measure mRNA levels of overexpressing LMP-1 and 18S, real-time PCR is performed using the TaqMan Real-Time PCR Kit (Applied Biosystems, USA). 25 ml of the reaction included 5 ml cDNA, 3.75 picomolar primers and 12.5 ml SYBR TaqMan PCR master mix (2x, Biorad, USA).

Real-time PCR is performed in three steps: 1 step between 50 parts at 2 ° C, 2 steps at 10 ° C at 95 ° C and 15 seconds at 95 ° C using GeneAmp (5700 Sequence Detection System, Applied Biosystems, USA), 60 It consists of three steps of 1 minute in a figure. PCR results are subjected to dislocation curve analysis. The critical cycle (Ct) of each reaction is normalized according to the 18S using the comparative "Ct method.

The standard curve for BMPs is the concentration of human BMP-2, 4, 6 and 7 (Genetics Institute, USA) dissolved in 0.05 mol / L bicarbonate buffer (0.1 ng / 100 μL per well to 1000 ng / 100 μL per well). Configure by increasing. 100 ml of the sample is added to each well. After overnight incubation at 4 ° C., three washes with saline (including 0.5% Tween 20: PBST) buffered with 0.01 M phosphate and the unreacted sites were blocked with 1% bobbin albumin (Sigma, USA) for 1 hour at room temperature. . Plates are washed with PBST and primary antibody (1: 1000) is added to each well with 100 microliter Ellicott and incubated for 2 hours at room temperature. Polyconal goth antibodies against BMP2, 4, and 6 (Santa Cruz Inc, USA) and rabbit antibodies against BMP-7 (Sigma, USA) are used. Plates are washed with PBST and incubated with alkaline phosphatase conjugate anti-goat IgG and anti-rabbit IgG (Sigma, USA) for 1 hour at room temperature. Discoloration of the substrate p-nitrophenyl phosphate (Sigma, USA) for 20 minutes before reaction is stopped by 3N NaOH. Discoloration is quantitatively determined by the difference in absorbance at 405 nm using an Elx800-microplate reader (Bio-Tek Instruments, USA).

To quantify the results, linear regression plots are made according to each standard. In all cases, the concentration of the sample is interpolated from the standard linear recreation plot according to the corresponding value at the same absorbance as reference.

BMP inhibition by nogin glycoproteins

Nogin is a glycoprotein that binds BMP-2, 4, 6, and 7 to prevent BMPs from activating cognate receptors. One form of mouse noggin (noggin-FC Sigma Chemical, USA) is used to determine the effect of blocking BMPs after AdLMP-1 treatment. Nogin is added at different concentrations (100, 200, 400, 800, 1600 and 3200 ng / ml), but on the first and third days after AdLMP-1 treatment. On day six, the medium is analyzed to test sGAG production using the DMMB method. The results show that LMP-1 effects are blocked by administration of Noggin, and that LMP-1 activity is partially related to the production of BMPs.

Claims (43)

A method of producing a cell permeable osteoinductive polypeptide comprising introducing the following expression construct into a suitable host cell: a) a polynucleotide encoding a cell permeable polypeptide; b) a polynucleotide that functionally binds to the cell permeable polypeptide and encodes the osteoinductive polypeptide such that the osteoinductive polypeptide is expressed as part of a fusion protein including the cell permeable polypeptide; c) a promoter located in direct transcription of the polynucleotide. The method of claim 1, wherein the expression construct also comprises a tablet tag. The method of claim 1, wherein the cell permeable polypeptide is selected from the group consisting of HIV-TAT, VP-22, saint factor signal peptide sequence, Pep-1, and Drosophila Antp peptide. The method of claim 1, wherein the cell permeable polypeptide is an HIV-TAT protein conversion domain. The method of claim 1, wherein the osteoinductive polypeptide is 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 method of claim 1, wherein the osteoinductive polypeptide is 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, method selected from the group of SEQ ID NO 8. A method of causing bone formation in a mammal comprising administering an effective amount of a fusion protein comprising a protein conversion domain and at least one osteoinductive polypeptide. 8. The method of claim 7, wherein the protein conversion domain is selected from the group consisting of HIV-TAT, VP-22, saint factor signal peptide sequence, Pep-1, and Drosophila Antp peptide. 8. The method of claim 7, wherein the protein conversion domain is an HIV-TAT protein conversion domain. The method of claim 7, wherein the osteoinductive polypeptide is 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 method of claim 7, wherein the osteoinductive polypeptide is 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, method selected from the group of SEQ ID NO 8. 8. The method of claim 7, wherein the fusion polypeptide is administered as an implant. 8. The method of claim 7, wherein the fusion polypeptide is administered as a hydrogel. 8. The method of claim 7, wherein the fusion polypeptide is administered through at least one multipotent progenitor cell. The method of claim 14, wherein the at least one multipotent progenitor cell is transplanted into a mammal. A polynucleotide encoding a fusion protein comprising a protein conversion domain and at least one osteoinductive polypeptide. The polynucleotide of claim 16, wherein the protein conversion domain is selected from the group consisting of HIV-TAT, VP-22, saint factor signal peptide sequence, Pep-1, and Drosophila Antp peptide. The polynucleotide of claim 16, wherein the protein conversion domain is an HIV-TAT protein conversion domain. The method of claim 16, wherein the osteoinductive polypeptide is 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 polynucleotides selected from the group. The method of claim 16, wherein the osteoinductive polypeptide is 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, polynucleotide selected from the group of SEQ ID NO 8. A method of inducing proteoglycans in a mammal comprising administering an effective amount of a fusion protein comprising a protein conversion domain and at least one osteoinductive polypeptide. The method of claim 21, wherein the protein conversion domain is selected from the group consisting of HIV-TAT, VP-22, saint factor signal peptide sequence, Pep-1, and Drosophila Antp peptide. The method of claim 21, wherein the protein conversion domain is an HIV-TAT protein conversion domain. The method of claim 21, wherein the osteoinductive polypeptide is 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, and BMP-7. The method of claim 21, wherein the osteoinductive polypeptide is 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, method selected from the group of SEQ ID NO 8. The method of claim 21, wherein the fusion polypeptide is administered as an implant. The method of claim 21, wherein the fusion polypeptide is administered through at least one multipotent progenitor cell. The method of claim 21, wherein the fusion polypeptide is administered through at least one multipotent progenitor cell. The method of claim 21, wherein the at least one multipotent progenitor cell is transplanted into a mammal. The method of claim 21, wherein the proteoglycan is agrecan. An isolated fusion polypeptide comprising a membrane-transfer peptide functionally linked to an osteoinductive polypeptide. The method of claim 31, wherein the membrane-transfer peptide is selected from the group consisting of HIV-TAT, VP-22, saint factor signal peptide sequence, Pep-1, and Drosophila Antp peptide. The method of claim 31, wherein the membrane-transfer peptide is an HIV-TAT protein conversion domain. The polypeptide of claim 31, wherein the osteoinductive polypeptide is 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 polypeptide of claim 31, wherein the osteoinductive polypeptide is 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, method selected from the group of SEQ ID NO 8. 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 conversion domain and at least one osteoinductive polypeptide. The method of claim 36, wherein the protein conversion domain is selected from the group consisting of HIV-TAT, VP-22, saint factor signal peptide sequence, Pep-1, and Drosophila Antp peptide. The method of claim 36, wherein the protein conversion domain is an HIV-TAT protein conversion domain. 37. The method of claim 36, wherein the osteoinductive polypeptide is 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. 37. The method of claim 36, wherein the osteoinductive polypeptide is 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, method selected from the group of SEQ ID NO 8. Bone selected from the group 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, SEQ ID NO 8, or a combination thereof Induction polypeptide. Osteoinduced polypeptides mixed in nucleic acid molecules complementary to the following sequences in standard state: tcctcatccg ggtcttgcat gaactcggtg. Osteoinduced polypeptide mixed in nucleic acid molecules complementary to the following sequences in a very stringent condition: gcccccgccc gctgacagcg ccccgcaa.

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