JP2005526488A - Methods for expressing LIM mineralized proteins in non-osseous cells - Google Patents

Abstract

Methods for expressing LIM mineralized proteins in non-osseous mammalian cells such as stem cells or intervertebral disc cells (eg, annulus fibrosus cells or nucleus pulposus cells) are described. The method involves transfecting a cell with an isolated nucleic acid comprising a nucleotide sequence encoding a LIM mineralized protein that is operably linked to a promoter. Transfection can be accomplished ex vivo or in vivo by direct injection of virus or naked DNA, or by non-viral vectors such as plasmids. LIM mineralized protein expression can stimulate proteoglycan and / or collagen production in cells capable of producing proteoglycan and / or collagen. A method of treating disc disease associated with trauma or disc degeneration is also described.

Description

This application claims priority to US Provisional Application No. 60 / 331,321, filed Nov. 14, 2001. This provisional application is fully incorporated herein by reference.
No. 09 / 124,238, filed Jul. 29, 1988, present U.S. Patent No. 6,300,127, and U.S. Patent Application No. 09 / 959,578, April 2000. Related to 28th application, pending. Each of these applications is fully incorporated herein by reference.

Background of the Invention
The field of the invention relates generally to methods for expressing LIM mineralized proteins in non-osteous cells such as intervertebral disc cells or nucleus pulposus cells. More specifically, the field of the invention relates to transfection of non-osteous cells such as intervertebral disc cells with nucleic acids encoding LIM mineralized proteins.

Technical background Osteoblasts are thought to differentiate from pluripotent mesenchymal stem cells. Osteoblast maturation results in the secretion of extracellular matrix that can mineralize and form bone. The control of this complex process is not well understood, but is thought to involve a group of signaling glycoproteins known as bone morphogenetic proteins (BMPs). These proteins have been shown to be involved in embryonic dorsal-ventral patterning, limb bud development, and fracture repair in adult animals. B. L. Hogan , Genes & Develop. , 10, 1580 (1996). This group of transforming growth factor-beta superfamily secreted proteins has diverse activities in diverse cell types at different stages of differentiation; differences in physiological activity between these closely related molecules are still It has not been elucidated. D. M.M. Kingsley , Trends Genet. , 10, 16 (1994).

In order to better recognize the unique physiological role of different BMP signaling proteins, we recently compared the potency of BMP-6 to induce rat calvarial osteoblast differentiation with that of BMP-2 and BMP-4 did. Boden et al. , Endocrinology, 137, 3401 (1996). We studied this process in the first passage (secondary) cultures of fetal rat calvaria that require BMP or glucocorticoid to initiate differentiation. In this model of forming membranous bone, glucocorticoid (GC) or BMP will initiate differentiation into mineralized bone nodules that can secrete osteocalcin, an osteoblast specific protein. This secondary culture system is different from primary rat osteoblast culture that undergoes spontaneous differentiation. In this secondary system, glucocorticoids produced a 10-fold induction of BMP-6 mRNA and protein expression, which was involved in promoting osteoblast differentiation. Boden et al. , Endocrinology, 138, 2920 (1997).

  In addition to extracellular signals such as BMP, intracellular signals or regulatory molecules may also play a role in the cascade of events leading to new bone formation. One broad class of intracellular regulatory molecules are LIM proteins, which are so named because they possess characteristic structural motifs known as LIM domains. The LIM domain is a cysteine-rich structural motif composed of two special zinc fingers linked by a two amino acid spacer. Some proteins have only a LIM domain, while others contain a variety of additional functional domains. LIM proteins form a diverse group, including transcription factors and cytoskeletal proteins. The main role of the LIM domain appears to mediate protein-protein interactions through the formation of dimers with the same or different LIM domains, or by binding to separate proteins.

  In LIM homeodomain proteins, ie proteins having both LIM domain and homeodomain sequences, the LIM domain functions as a negative regulatory element. Although LIM homeodomain proteins are involved in the regulation of cell lineage determination and differentiation control, LIM-only proteins may have a similar role. LIM-only proteins are also involved in the regulation of cell proliferation because several genes encoding such proteins are involved in oncogenic chromosomal translocation.

  Humans and other mammalian species are prone to diseases or injuries that require bone repair and / or bone regeneration processes. For example, the treatment of fractures may be improved by new treatment measures that can stimulate the natural bone repair mechanism and thereby reduce the time it takes for the fractured bone to heal. In another example, an individual suffering from a systemic bone disorder such as osteoporosis will benefit from a therapeutic procedure that will result in the systemic formation of new bone. Such treatment measures will reduce the incidence of fractures due to the loss of bone mass characteristic of the disease.

  For at least these reasons, extracellular factors such as BMP have been studied for the purpose of using them to stimulate the formation of new bone in vivo. Despite the initial success achieved with BMP and other extracellular signaling molecules, their use was associated with several disadvantages. For example, increasing the production of new bone requires a relatively large amount of purified BMP, thereby increasing the cost of such therapies. Furthermore, extracellular proteins are susceptible to degradation after introduction into a host animal. Furthermore, since they are typically immunogenic, there is always the possibility of stimulating an immune response against the administered protein.

  Because of these concerns, it would be desirable to have an available treatment that uses intracellular signaling molecules to induce new bone formation. Due to advances in the field of gene therapy, nucleotide fragments encoding intracellular signals responsible for part of the bone formation process are now introduced into osteogenic progenitor cells, ie cells involved in bone formation, or peripheral blood leukocytes Is possible. Gene therapy for bone formation provides several potential advantages: (1) lower production costs; (2) extracellular signals have the ability to achieve extended expression More effective compared to therapeutic measures; (3) avoiding the possibility that treatment with extracellular signals may be hindered because there are only a limited number of receptors for extracellular signals. (4) enables the direct delivery of potential transfected osteoprogenitor cells to the site in need of localized bone formation; and (5) enables systemic bone formation; Would provide treatment for osteoporosis and other metabolic bone diseases.

  In addition to bone disease, humans and other mammalian species are also prone to intervertebral disc degeneration, which is involved, inter alia, in lower back pain, disc herniation, and spinal stenosis. Intervertebral disc degeneration is associated with progressive loss of the proteoglycan matrix. This may make the intervertebral disc susceptible to biomechanical injury and degeneration. Thus, it would be desirable to have a method of stimulating proteoglycan and / or collagen synthesis by appropriate cells, such as nucleus pulposus cells, annulus fibrosus cells, and intervertebral disc cells.

SUMMARY OF THE INVENTION According to a first aspect of the present invention, a method for expressing a LIM mineralized protein in non-osseous mammalian cells is provided. In accordance with this aspect of the invention, the method comprises transfecting a cell with an isolated nucleic acid comprising a nucleotide sequence encoding a LIM mineralized protein operably linked to a promoter. The cell can be a cell capable of producing proteoglycan and / or collagen such that expression of the LIM mineralized protein stimulates proteoglycan and / or collagen synthesis in the cell. An isolated nucleic acid according to this aspect of the invention is a nucleic acid capable of hybridizing under standard conditions to a nucleic acid molecule complementary to the full length of SEQ ID NO: 25; and / or under highly stringent conditions It can be a nucleic acid molecule capable of hybridizing to a nucleic acid molecule complementary to the full length of number 26. The cells can be stem cells, intervertebral disc cells, annulus fibrosus cells, or nucleus pulposus cells.

  According to a second aspect of the present invention, there is provided a non-osseous mammalian cell comprising an isolated nucleic acid sequence encoding a LIM mineralized protein. In accordance with this aspect of the invention, the cells can be stem cells, nucleus pulposus cells, annulus fibrosus cells, or intervertebral disc cells.

  According to a third aspect of the present invention, a method of treating an intervertebral disc injury or disease is provided. According to this aspect of the invention, the method comprises transfecting the isolated nucleic acid into a mammalian cell capable of producing proteoglycan and / or collagen. The isolated nucleic acid comprises a nucleotide sequence encoding a LIM mineralized protein that is operably linked to a promoter. LIM mineralized proteins stimulate proteoglycan and / or collagen synthesis in cells.

  According to a fourth aspect of the present invention, an intervertebral disc implant is provided. In accordance with this aspect of the invention, the implant comprises a plurality of mammalian cells comprising a carrier material and an isolated nucleic acid sequence encoding a LIM mineralized protein. Again according to this aspect of the invention, the carrier material comprises a porous matrix of biocompatible material and the mammalian cells are incorporated into the carrier material.

Detailed Description of Preferred Embodiments The present invention relates to transfection of non-osteous cells using nucleic acids encoding LIM mineralized proteins. The inventors of the present invention can increase the synthesis of proteoglycans, collagen, and other intervertebral disc components and tissues as a result of transfection of non-osteous cells such as intervertebral disc cells with nucleic acids encoding LIM mineralized proteins. I discovered that. The present invention also provides a method of treating intervertebral disc disease associated with loss of proteoglycan, collagen, or other intervertebral disc components.

Both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further description of the invention as claimed.
Abbreviations and definitions

  The LIM gene (10-4 / RLMP) was isolated from stimulated rat calvarial osteoblast cultures (SEQ ID NO: 1, SEQ ID NO: 2). See U.S. Patent No. 6,300,127. This gene has been cloned, sequenced, and assayed for its ability to enhance bone mineralization effects in vitro. The protein RLMP has been found to affect the differentiation of cells into the osteoblast lineage as well as mineralization of the bone matrix. Unlike other known cytokines (eg BMP), RLMP is not a secreted protein but an intracellular signaling molecule. This feature has the advantage of providing easier evaluation of the transfected cells along with intracellular signaling amplification. This is also more effective and suitable for specific in vivo applications. Suitable clinical applications include fractures, bone defects, enhanced bone repair in bone grafts, and normal homeostasis in patients with osteoporosis.

  The amino acid sequence of the corresponding human protein, termed human LMP-1 (“HLMP-1”), has also been cloned, sequenced and deduced. See U.S. Patent No. 6,300,127. Human proteins have been found to show enhanced bone mineralization effects in vitro and in vivo.

  In addition, a truncated (short) version of HLMP-1 termed HLMP-1s has been characterized. See U.S. Patent No. 6,300,127. This short version results from a point mutation in one source of the cDNA clone and provides a stop codon that partially excises the protein. HLMP-1s has been found to be fully functional when expressed in cell culture and in vivo.

  Using PCR analysis of a human heart cDNA library, two alternative splicing variants (referred to as HLMP-2 and HLMP-3) have been identified, which in the nucleotide sequence encoding HLMP-1, It differs from HLMP-1 in the region between base pairs 325-444. See US patent application Ser. No. 09 / 959,578, filed Apr. 28, 2000, pending. The HLMP-2 sequence has a 119 base pair deletion and a 17 base pair insertion in this region. Compared to HLMP-1, there is no deletion in the nucleotide sequence encoding HLMP-3, but the sequence has the same 17 base pairs as HLMP-2, which is at position 444 of the HLMP-1 sequence. Has been inserted.

  LMPs are pluripotent molecules that control or affect several biological processes. Different splicing variants of LMP are expected to have different biological functions in mammals. They can play a role in the growth, differentiation and / or regeneration of various tissues. For example, some types of LMP are expressed not only in bone, but also in muscle, tendon, ligament, spine, peripheral nerve, and cartilage.

  According to one aspect, the present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding a LIM mineralized protein operably linked to a promoter; said mammalian nucleic acid capable of producing proteoglycan said Method of stimulating proteoglycan and / or collagen synthesis in mammalian cells by transfecting an isolated nucleic acid sequence; and expressing said nucleotide sequence encoding a LIM mineralized protein, thereby stimulating proteoglycan synthesis About. Mammalian cells can be non-osseous cells such as intervertebral disc cells, annulus fibrosus cells, or nucleus pulposus cells. Transfection can occur either ex vivo or in vivo by direct injection of virus or naked DNA, eg, a plasmid. In a particular embodiment, the virus is a recombinant adenovirus, preferably AdHLMP-1.

  Another aspect of the invention comprises non-osseous mammalian cells comprising an isolated nucleic acid sequence encoding a LIM mineralized protein. Non-osseous mammalian cells can be stem cells (eg, pluripotent stem cells or mesenchymal stem cells) or intervertebral disc cells, preferably nucleus pulposus cells or annulus fibrosus cells.

  In a different aspect, the present invention provides a method for expressing an isolated nucleotide sequence encoding a LIM mineralized protein in non-osseous mammalian cells, wherein the LIM mineralized protein is operably linked to a promoter. Providing an isolated nucleic acid comprising a coding nucleotide sequence; transfecting the isolated nucleic acid sequence into a non-boneed mammalian cell; and expressing the nucleotide sequence encoding a LIM mineralized protein. And relates to the method. Non-osseous mammalian cells can be stem cells or intervertebral disc cells (eg, nucleus pulposus cells or annulus fibrosus cells). Transfection can occur either ex vivo or in vivo by direct injection of virus or naked DNA, eg, a plasmid. The virus can be a recombinant adenovirus, preferably AdHLMP-1.

  In yet another aspect, the invention relates to a method of treating intervertebral disc disease by reversing, interfering with or delaying intervertebral disc degeneration, wherein the LIM mineralized protein is operably linked to a promoter. An isolated nucleic acid comprising a nucleotide sequence that encodes; a mammalian cell capable of producing proteoglycans is transfected with the isolated nucleic acid sequence; and the nucleotide sequence encoding a LIM mineralized protein is expressed. In particular, by stimulating proteoglycan synthesis in the cells, thereby reversing, stopping or delaying the degeneration of the disc. Intervertebral disc disease may involve lower back pain, disc herniation, or spinal stenosis. Mammalian cells can be non-osseous cells such as stem cells or intervertebral disc cells (eg, nucleus pulposus cells or annulus fibrosus cells).

  Transfection can occur either ex vivo or in vivo by direct injection of virus or naked DNA, eg, a plasmid. In a particular embodiment, the virus is a recombinant adenovirus, preferably AdHLMP-1.

  The present invention relates to a novel mammalian LIM protein, referred to herein as LIM mineralized protein, or LMP. The present invention more particularly relates to a human LMP known as HLMP or HLMP-1, or an alternative splicing variant of human LMP known as HLMP-2 or HLMP-3. Applicants of the present invention have discovered that these proteins enhance bone mineralization in mammalian cells grown in vitro. When produced in mammals, LMP also induces bone formation in vivo.

  Bone marrow cells, osteogenic progenitor cells, peripheral blood cells, and stem cells (eg, pluripotent stem cells or mesenchymal stem cells) are ex vivo transfected with a nucleic acid encoding a LIM mineralized protein (eg, LMP or HLMP), and then Reimplanting transfected cells into a donor is suitable for treating a variety of bone related disorders or injuries. For example, using this method: increasing fracture repair; generating bone in segmental defects; providing bone graft substitutes for fractures; promoting tumor remodeling or spinal fixation; and weak bones Alternatively, it may be possible to provide local treatment (by injection) of osteoporotic bone, such as in osteoporosis of the hips, vertebrae, or wrists. Transfection with LMP or HLMP-encoding nucleic acids also: accelerates repair of fractured long bones, percutaneous injection of transfected bone marrow cells; delayed healing or failure of long bone fractures, or spinal fusion pseudo-joints It is also useful for treatment; and new bone induction in avascular necrosis of the hip or knee.

In addition to ex vivo methods of gene therapy, transfection of recombinant DNA vectors comprising nucleic acid sequences encoding LMP or HLMP can be achieved in vivo. When the DNA fragment encoding LMP or HLMP is inserted into an appropriate viral vector, such as an adenoviral vector, the viral construct can be directly injected into a body site where bone formation within the cartilage is desired. Directly using subcutaneous injections to introduce LMP or HLMP sequences, to obtain bone marrow cells (for ex vivo transfection), or to re-add them to the site where the patient's new bone is needed. For transplantation, stimulation of bone formation can be achieved without the need for surgical intervention. Alden et al. , Neurological Focus (1998) demonstrated the utility of a direct injection method of gene therapy using cDNA encoding BMP-2 cloned into an adenoviral vector.

In vivo gene therapy can also be performed by direct injection of a recombinant plasmid comprising a nucleic acid sequence encoding HLMP, naked or unencapsulated, at an appropriate body site. In this aspect of the invention, transfection occurs when naked plasmid DNA is taken up or internalized by the appropriate target cell described. As in in vivo gene therapy with viral constructs, direct injection of naked plasmid DNA offers the advantage that little or no surgical intervention is required. Direct gene therapy with naked plasmid DNA encoding the endothelial cell mitogen, VEGF (vascular endothelial growth factor) has been successful in human patients. Baumagartner et al. , Circulation, 97, 12, 1114-1123 (1998).

  For intervertebral disc applications, this can be accomplished by harvesting cells from the intervertebral disc, transfecting the cells in vitro with a nucleic acid encoding LIM, and then introducing the cells into the intervertebral disc. For example, cells can be harvested from the disc or reintroduced into the disc using any means known to those skilled in the art, such as any suitable surgical technique for use with the spine. is there. In one embodiment, the cells are introduced into the disc by injection.

  Again according to the present invention, stem cells (eg pluripotent stem cells or mesenchymal stem cells) can be transfected ex vivo and introduced into the intervertebral disc (eg by injection) with a nucleic acid encoding a LIM mineralized protein It is.

Cells transfected ex vivo can also be combined with a carrier to form a disc implant. Thereafter, a carrier comprising the transfected cells can be implanted into the subject's disc. Suitable carrier materials are described in Helm et al. , “Bone Graft Substitutes for the Promotion of Spinal Arthrosis”, Neurosurg Focus, Vol. 10 (4): disclosed in April 2001. The carrier preferably comprises a biocompatible porous matrix such as a demineralized bone matrix (DBM), a biocompatible synthetic polymer matrix or a protein matrix. Suitable proteins include extracellular matrix proteins such as collagen. Prior to transplantation, cells transfected with LMP ex vivo can be incorporated into a carrier (ie, into the pores of a porous matrix).

  Similarly, for intervertebral disc applications where cells are transfected in vivo, DNA can be introduced into the intervertebral disc using any suitable method known to those skilled in the art. In one embodiment, the nucleic acid is injected directly into the intervertebral space.

  Transient expression of LMP is achieved by delivering LMP to osteogenic cells using an adenoviral vector. This occurs because adenovirus is not incorporated into the genome of the target cell being transfected. Transient expression of LMP, ie expression that occurs during the lifetime of the transfected target cell, is sufficient to achieve the objectives of the present invention. However, stable expression of LMP can occur when a vector that is incorporated into the genome of the target cell is used as a delivery vehicle. For example, vectors based on retroviruses are suitable for this purpose.

  Stable expression of LMP is particularly useful for treating a variety of systemic bone related disorders such as osteoporosis and osteogenesis imperfecta. For this aspect of the invention, in addition to using a vector integrated into the genome of the target cell to deliver the nucleotide sequence encoding LMP to the target cell, it is placed under the control of a promoter capable of controlling LMP expression. Is possible. For example, a promoter that is switched on by exposure to an exogenous inducing agent such as tetracycline is suitable.

  Using this approach, it is possible to stimulate the formation of new bone on a systemic scale by administering an effective amount of an exogenous inducer. Once a sufficient amount of bone has been achieved, administration of the exogenous inducer can be discontinued. This process can be repeated as needed, for example to replace bone loss resulting from osteoporosis. Antibodies specific for HLMP are particularly suitable for use in a method of assaying the osteoinductive potential of patient cells, ie the osteogenic potential. In this way, it is possible to identify patients at risk of slow or inferior bone repair healing. HLMP-specific antibodies are also suitable for use in marker assays to identify risk factors in bone degenerative diseases such as osteoporosis.

  The gene of the present invention is prepared by ligating a nucleic acid segment encoding LMP to other nucleic acid sequences, such as cloning vectors and / or expression vectors, according to well-known methods and conventional methods. The methods necessary to construct and analyze these recombinant vectors have been described, such as restriction endonuclease digestion, cloning protocols, mutagenesis, oligonucleotide organic synthesis and DNA sequencing. The dideoxy terminator method is preferred for DNA sequencing.

Many technical books on recombinant DNA methods have been published, including Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, (1988), Davis et al. , Basic Methodology. , Elsevier (1986) and Ausubel et al. , Current Protocols in Molecular Biology, Wiley Interscience (1988). These reference manuals are specifically incorporated herein.

Amplification of the DNA or cDNA indicated by the primer is a common step in gene expression of the present invention. This is typically done by polymerase chain reaction (PCR). PCR is described in US Pat. No. 4,800,159 to Mullis et al . And other publication sources. The basic principle of PCR is the exponential replication of DNA sequences with a continuous period of primer extension. The extension product of one primer becomes a template for another nucleic acid molecule synthesis when hybridized with another primer. The primer-template complex acts as a substrate for DNA polymerase that extends the primer when performing a replication function. A conventional enzyme applied to PCR is a thermostable DNA polymerase isolated from Thermus aquaticus, or Taq DNA polymerase.

  There are many variations of the basic PCR method, and the particular method chosen in any of the predetermined steps necessary to construct the recombinant vector of the invention is readily performed by those skilled in the art. For example, to measure cellular expression of 10-4 / RLMP, RNA is extracted and reverse transcribed under standard and well-known methods. The resulting cDNA is then analyzed for the appropriate mRNA sequence by PCR.

  A gene encoding a LIM mineralized protein is expressed in an expression vector of a recombinant expression system. Of course, the constructed sequence does not have to be the same as the original sequence or its complementary sequence, and any sequence that expresses LMP having osteogenic activity despite being determined by the degeneracy of the DNA code. It is possible that Other modifications such as conservative amino acid substitutions or the presence of an amino terminal methionine residue can also be used.

  A ribosome binding site active in the selected host expression system is ligated to the 5 'end of the chimeric LMP coding sequence to form a synthetic gene. By ligating to an appropriately linearized plasmid, it is possible to insert the synthetic gene into any one of a great variety of vectors for expression. Regulatable promoters, such as the E. coli lac promoter, are also suitable for expression of the chimeric coding sequence. Other suitable regulatable promoters include trp, tac, recA, T7 and the lambda promoter.

  Stable transformation by transfecting recipient cells with DNA encoding LMP by one of several standard published methods such as calcium phosphate precipitation, DEAE-dextran, electroporation or protoplast fusion. Form the body. Calcium phosphate precipitation is preferred, especially when performed as follows.

Before transferring the DNA to the cells, it is precipitated with calcium phosphate according to the method of Graham and Van Der , Virology, 52, 456 (1973). An aliquot of 40-50 μg of DNA is used for 0.5 × 10 ⁶ cells in a 100 mm dish with salmon sperm DNA or bovine thymus DNA as a carrier. The DNA was mixed with 0.5 ml of 2X Hepes solution (280 mM NaCl, 50 mM Hepes and 1.5 mM Na ₂ HPO ₄ , pH 7.0) to which an equal volume of ₂ × CaCl ₂ (250 mM CaCl ₂ and 10 mM Hepes, pH 7.0). ) Is added. The white granular precipitate that appears after 30-40 minutes is evenly distributed drop by drop on the cells and is incubated at 37 ° C. for 4-16 hours. The medium is removed and the cells are shocked with 15% glycerol in PBS for 3 minutes. After removal of glycerol, the cells are fed Dulbecco's minimal essential medium (DMEM) containing 10% fetal calf serum.

DNA has also been described by: Kimura et al. , Virology, 49: 394 (1972) and Somepayrac et al ., Proc. Natl. Acad. Sci. USA, 78, 7575 (1981) DEAE-dextran method; Potter , Proc. Natl. Acad. Sci. USA, 81, 7161 (1984); and Sandri-Goddin et al ., Molec. Cell. Biol. 1, 743 (1981) can also be used for transfection.

  Solid phase phosphoramidite chemistry is the preferred method for organic synthesis of oligodeoxynucleotides and polydeoxynucleotides. In addition, many other organic synthesis methods are available. These methods are readily adapted by those skilled in the art to the specific sequences of the invention.

The invention also includes nucleic acid molecules that hybridize under standard conditions to any of the nucleic acid sequences encoding the LIM mineralized proteins of the invention. “Standard hybridization conditions” vary with probe size, nucleic acid reagent background and concentration, as well as hybridization type, eg, in situ, Southern blot, or DNA-RNA hybrid hybridization (Northern blot). I will. The determination of “standard hybridization conditions” is within the level of the art. See, for example, US Pat. No. 5,580,775 to Fremeau et al. , Incorporated herein for this purpose. Southern , J. et al. 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, 7.19-1.50 (1989).

One preferred set of standard hybridization conditions is 50% formamide, 5XSSPE (150 nM NaCl, 10 mM NaH ₂ PO ₄ [pH 7.4], 1 mM EDTA [pH 8.0]), 5 × Denhardt's solution (for 100 ml water). 20 mg Ficoll, 20 mg polyvinylpyrrolidone and 20 mg BSA) in 10% dextran sulfate, 1% SDS and 100 μg / ml salmon sperm DNA for 2 hours at 42 ° C. ³² P-labeled cDNA probe is added and hybridization is continued for 14 hours. The blot is then washed twice for 20 minutes at 2XSSPE, 0.1% SDS, 22 ° C, followed by 1 hour at 65 ° C in 0.1XSSPE, 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 “very stringent conditions”, a probe will hybridize to a target sequence if the probe and its target sequence are substantially identical. As in the case of standard hybridization conditions, one skilled in the art will determine the conditions under which only substantially identical sequences will hybridize, taking into account the level of skill in the art and the nature of the particular experiment. Is possible.

  In accordance with one aspect of the invention, an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a LIM mineralized protein is provided. A nucleic acid molecule according to the present invention hybridizes under standard conditions to a nucleic acid molecule complementary to the full length of SEQ ID NO: 25 and / or complements to the full length of SEQ ID NO: 26 under very stringent conditions. It can be a molecule that hybridizes to a typical nucleic acid molecule. More specifically, an isolated nucleic acid molecule according to the present invention can encode HLMP-1, HLMP-1s, RLMP, HLMP-2, or HLMP-3.

Another aspect of the invention includes proteins encoded by nucleic acid sequences. In yet another aspect, the invention relates to the identification of such proteins based on anti-LMP antibodies. In this embodiment, protein samples are prepared for Western blot analysis by lysing the cells and separating the proteins by SDS-PAGE. Proteins are transferred to nitrocellulose by electroblotting as described in Ausubel et al. , Current Protocols in Molecular Biology, John Wiley and Sons (1987). After blocking the filter with instant skim milk powder (1 gram in 100 ml PBS), anti-LMP antibody is added to the filter and incubated for 1 hour at room temperature. Filters are washed thoroughly with phosphate buffered saline (PBS) and incubated with horseradish peroxidase (HRPO) -antibody conjugate for 1 hour at room temperature. Again wash the filter thoroughly with PBS and identify the antigen band by adding diaminobenzidine (DAB).

Monospecific antibodies are the reagents selected in the present invention and are used specifically to analyze patient cells for specific properties associated with LMP expression. A “monospecific antibody” is defined herein as a single antibody species or multiple antibody species with homogeneous binding properties for LMP. “Homogeneous binding” as used herein refers to the ability of an antibody species to bind to a particular antigen or epitope, such as that associated with LMP. Monospecific antibodies to LMP are purified from mammalian antisera containing antibodies that are reactive to LMP, or as monoclonal antibodies that are reactive with LMP using Kohler and Milstein techniques. Prepared. Kohler et al. , Nature, 256, 495-497 (1975). For example, LMP-specific antibodies are made by immunizing animals such as mice, rats, guinea pigs, rabbits, goats or horses with an appropriate concentration of LIM, either with or without an immune adjuvant.

  In this process, preimmune serum is collected prior to the first immunization. Each animal receives between about 0.1 mg and about 1000 mg of LMP, if desired, associated with an acceptable immune adjuvant. Such acceptable adjuvants include, but are not limited to, Freund's complete adjuvant, Freund's incomplete adjuvant, alum sediment, water-in-oil emulsion containing Corynebacterium parvum and tRNA adjuvant. Is included. The initial immunization consists of LMP injected at multiple sites, preferably subcutaneously (SC), intraperitoneally (IP) or both, preferably in Freund's complete adjuvant. Antibody titers are measured by bleeding from each animal at regular intervals, preferably weekly. The animal may or may not receive a booster injection after the initial immunization. Animals receiving booster injections are generally given equal amounts of antigen in Freund's incomplete adjuvant by the same route. Booster injections are given approximately every 3 weeks until the maximum titer is obtained. Approximately 7 days after each booster immunization, or after a single immunization, animals are bled approximately weekly, serum is collected, and aliquots are stored at approximately -20 ° C.

A monoclonal antibody (mAb) that is reactive with LMP is prepared by immunizing inbred mice, preferably Balb / c mice, with LMP. As discussed above, from about 0.1 mg to about 10 mg, preferably about 1 mg of LMP in about 0.5 mg of buffer or saline in an equal volume of an acceptable adjuvant, by the IP or SC route. Immunize mice. Freund's complete adjuvant is preferred. Mice are primed on day 0 and rested for about 3 to 30 weeks. Immunized mice are given one or more boosters of about 0.1 to about 10 mg of LMP in a buffer solution such as phosphate buffered saline by the intravenous (IV) route. Lymphocytes from antibody positive mice, preferably splenic lymphocytes, are obtained by removing the spleen from immunized mice by standard methods known in the art. Hybridoma cells are produced by mixing splenic lymphocytes with a suitable fusion partner, preferably myeloma cells, under conditions that will allow stable hybridoma formation. Fusion partners include, but are not limited to: mouse myeloma P3 / NS1 / Ag4-1; MPC-11; S-194 and Sp2 / 0, with Sp2 / 0 being preferred. Antibody-producing cells and myeloma cells are fused in polyethylene glycol at a concentration of about 1,000 molecular weight and about 30% to about 50%. Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin in fortified Dulbecco's modified Eagle medium (DMEM) by methods known in the art. Approximately 14 days, 18 days, and 21 days, supernatants are collected from growth positive wells and used for antibody production by immunoassays such as solid phase immunoradioactivity assay (SPIRA) using LMP as an antigen. Screen. The culture is also tested in an octalony precipitation assay to determine the mAb isotype. MacPherson , "Soft Agar Techniques: Tissue Culture Methods and Applications", Krue and Paterson (supervised), cloning of cells from positive cells by means of hybrid technology such as Agaric Press (1973). See also Harlow et al. , Antibodies: A Laboratory Manual, Cold Spring Laboratory (1988).

Monoclonal antibodies can also be produced in vivo by injecting approximately 0.5 ml per mouse of about 2 × 10 ⁶ to about ⁶ × 10 ⁶ hybridoma cells about 4 days after priming to Balb / c mice primed with pristane. Approximately 8-12 days after cell transfer, ascites fluid is collected and monoclonal antibodies are purified by techniques known in the art.

  In vitro production of anti-LMP mAbs is performed by growing hybridoma cell lines in DMEM containing about 2% fetal bovine serum to obtain sufficient amounts of specific mAbs. The mAb is purified by techniques known in the art.

  Ascites or hybridoma cultures by various serological or immunological assays, including but not limited to sedimentation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technology and radioimmunoassay (RIA) technology Determine antibody titer of fluid. Similar assays are used to detect the presence of LMP in body fluids or tissues and cell extracts.

  It is possible to produce antibodies specific for LMP polypeptide fragments, full length nascent LMP polypeptides, or variants or alleles thereof using the methods described above for producing monospecific antibodies. It will be readily apparent to the merchant.

  In another aspect, the present invention relates to alternative splicing variants of HLMP-1. PCR analysis of human heart cDNA reveals two HLMP alternatively spliced variant mRNAs, designated HLMP-2 and HLMP-3, which are between base pairs 325-444 of the HLMP-1 sequence. The region is different from HLMP-1. The HLMP-2 sequence has a 119 base pair deletion and a 17 base pair insertion in this region. These changes preserve the reading frame and result in a 423 amino acid protein that has a net loss of 34 amino acids compared to HLMP-1 (6 amino acids are inserted in addition to the 40 amino acid deletion). ) HLMP-2 contains a C-terminal LIM domain present in HLMP-1.

  Compared to HLMP-1, HLMP-3 has no deletion but has the same 17 base pair insertion at position 444. This insertion shifts the reading frame, resulting in a stop codon at base pairs 459-461. As a result, HLMP-3 encodes a 153 amino acid protein. This protein lacks the C-terminal LIM domain present in HLMP-1 and HLMP-2. The expected size of the proteins encoded by HLMP-2 and HLMP-3 was confirmed by Western blot analysis.

  PCR analysis of the tissue distribution of the three splicing variants revealed that they were differentially expressed, with different isoforms predominant in different tissues. HLMP-1 appears to be the major type expressed in leukocytes, spleen, lung, placenta, and fetal liver. HLMP-2 appears to be the major isoform of skeletal muscle, bone marrow, and heart tissue. However, HLMP-3 was not the major isoform in any of the tissues examined.

  Overexpression of HLMP-3 in secondary rat osteoblast cultures resulted in bone nodule formation (287 ± 56) similar to the effects seen with glucocorticoids (272 ± 7) and HLMP-1 (232 ± 200). Induced. Since HLMP-3 lacks a C-terminal LIM domain, this region is not required for osteoinductive activity.

  However, overexpression of HLMP-2 did not induce nodule formation (11 ± 3). These data suggest that the deleted 119 base pair encoded amino acid is required for bone induction. The data also suggests that the distribution of HLMP splicing variants may be important for tissue specific functions. Surprisingly, we have shown that HLMP-2 inhibits steroid-induced osteoblast formation in secondary rat osteoblast cultures. Therefore, HLMP-2 may have therapeutic utility in clinical situations where bone formation is not desirable.

  July 22, 1997, a sample of 10-4 / RLMP (vector pRc / CMV2 containing insert 10-4 clone / RLMP) in a vector designated pCMV2 / RLMP, American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, MD 20852. The culture deposit number for this deposit is 209153. On March 19, 1998, a sample of vector pHis-A containing the insert HLPM-1s was deposited with the American Type Culture Collection (“ATCC”). The culture deposit number for this deposit is 209698. April 14, 2000, plasmids pHAhLMP-2 (vector pHisA containing cDNA insert derived from human heart muscle cDNA of HLMP-2) and pHAhLMP-3 (vector pHisA containing cDNA insert derived from human heart muscle cDNA of HLMP-3) ) Samples under the conditions of the Budapest Treaty, ATCC, 10801 University Blvd. , Manassas, VA, 20110-2209, USA. The deposit numbers for these deposits are PTA-1698 and PTA-1699, respectively. These deposits will be maintained by the ATCC for at least 30 years as required by the Budapest Treaty and will be made publicly available when patents disclosing them are granted. The availability of deposits does not constitute a license to implement the present invention in the impairment of patent rights granted by governmental legal action.

Enzyme assays, protein purification, and other conventional biochemical methods are used in evaluating the nucleic acids, proteins, or antibodies of the present invention. DNA and RNA are analyzed by Southern and Northern blotting techniques, respectively. Typically, the sample to be analyzed is size fractionated by gel electrophoresis. Thereafter, the DNA or RNA in the gel is transferred to a nitrocellulose membrane or a nylon membrane. A blot, which is a replica of the sample pattern in the gel, was then hybridized with the probe. Typically, the probe is radiolabeled, preferably with ³² P, but it is possible to label the probe with other signal generating molecules known in the art. Thereafter, the specific band of interest can be visualized by a detection system such as autoradiography.

  For the purpose of illustrating preferred embodiments of the invention, the following non-limiting examples are included. These results demonstrate the feasibility of inducing or enhancing bone formation using the LIM mineralized proteins of the present invention and the isolated nucleic acid molecules encoding these proteins.

Example 1: Calvarial cell culture As described above, rat calvarial cells, also known as rat osteoblasts ("ROB"), were obtained from rats 20 days prior to delivery. Boden et al. , Endocrinology, 137, 8, 3401-3407 (1996). Primary cultures were grown to confluence (7 days), trypsinized, and passaged as primary subculture cells to 6-well plates (1 × 10 ⁵ cells / 35 mm well). Subcultured cells that were confluent on day 0 were grown for an additional 7 days. Starting on day 0, every 3 or 4 days, medium was changed and treatment (Trm and / or BMP) was applied under lamina flow hood. The standard culture protocol was as follows: Day 1-7, MEM, 10% FBS, 50 μg / ml ascorbic acid, ± stimulation; Day 8-14, BGJb medium, 10% FBS, 5 mM β-GlyP (as a source of inorganic phosphate allowing mineralization). End point analysis of bone nodule formation and osteocalcin secretion was performed on day 14. Based on pilot experiments in this system, the BMP dose was selected as 50 ng / ml, which was the dose that showed an effect in the middle range of the dose-response curve for all BMPs studied.

Example 2: To investigate the potential functional role of LMP-1 during antisense treatment and cell culture membranous bone formation, antisense oligonucleotides that block LMP-1 mRNA translation were synthesized and glucocorticoids Secondary osteoblast cultures undergoing differentiation initiated by were processed. RLMP expression using a very specific antisense oligonucleotide (no significant homology to known rat sequences), corresponding to a 25 bp sequence (SEQ ID NO: 42) across the putative translation start site Inhibition was achieved. Control cultures received no oligonucleotide or sense oligonucleotide. Experiments were performed in the presence (preincubation) and absence of lipofectamine. Briefly, 22 μg of sense or antisense RLMP oligonucleotide was incubated in MEM for 45 minutes at room temperature. Following this incubation, either additional MEM or preincubated Lipofectamine / MEM (7% v / v; incubated for 45 minutes at room temperature) was added to bring the oligonucleotide concentration to 0.2 μM. The resulting mixture was incubated at room temperature for 15 minutes. The oligonucleotide mixture was then mixed with the appropriate medium, ie MEM / ascorbate / ± Trm, to achieve a final oligonucleotide concentration of 0.1 μM.

Cells were incubated with the appropriate medium (± stimulation) in the presence or absence of the appropriate oligonucleotide. The cultures initially incubated with Lipofectamine were re-fed with media containing neither Lipofectamine nor oligonucleotide after 4 hours of incubation (37 ° C .; 5% CO ₂ ). All cultures, particularly those dosed with oligonucleotides, were re-fed every 24 hours to maintain oligonucleotide levels.

  LMP-1 antisense oligonucleotides inhibited mineralized nodule formation and osteocalcin secretion in a dose-dependent manner, similar to the effects of BMP-6 oligonucleotides. LMP-1 antisense blockade in osteoblast differentiation cannot be rescued by addition of exogenous BMP-6, while BMP-6 antisense oligonucleotide inhibition was reversed by addition of BMP-6. This experiment further confirmed that LMP-1 was in an upstream position in the osteoblast differentiation pathway compared to BMP-6. LMP-1 antisense oligonucleotide also inhibited spontaneous osteoblast differentiation in primary rat osteoblast cultures.

Example 3: Quantification of mineralized bone nodule formation ROB cultures prepared according to Examples 1 and 2 were fixed overnight in 70% ethanol and stained with von Kossa silver stain. A semi-automated computerized video image analysis system was used to quantify the nodule number and nodule area in each well. Boden et al. , Endocrinology, 137, 8, 3401-3407 (1996). Thereafter, these values were divided to calculate the area value per nodule. This automated process was validated against a manual counting technique and proved a correlation coefficient of 0.92 (p <0.000001). All data are expressed as mean ± standard error of the mean (SEM), calculated from 5 or 6 wells for each condition. Each experiment was confirmed at least twice using cells from different calvarial preparations.

Example 4: Quantification of osteocalcin secretion
Use monospecific polyclonal antibodies (Pab) generated in our laboratory against the C-terminal nonapeptide of rat osteocalcin as described in Nanes et al. , Endocrinology, 127: 588 (1990). A competitive radioimmunoassay was used to measure osteocalcin levels in the medium. Briefly, 1 μg of nonapeptide was iodinated with 1 mCi ¹²⁵ I-Na by the lactoperoxidase method. Tubes from 200 ml of assay buffer (0.02 M sodium phosphate, 1 mM EDTA, 0.001% thimerosal, 0.025% BSA) from cell cultures in assay buffer at 100 g / tube. Medium or osteocalcin standard (0-12,000 fmol) was added. Then, Pab (1: 40,000; 100 μl) was added, followed by the addition of iodinated peptide (12,000 cpm; 100 μl). Samples to be tested for non-specific binding were prepared similarly, but without antibody.

  Bound and free PAb were separated by adding 700 μl goat anti-rabbit IgG followed by incubation at 4 ° C. for 18 hours. After centrifuging the sample at 1200 rpm for 45 minutes, the supernatant was decanted and the precipitate was counted in a gamma counter. Osteocalcin values were reported in fmol / 100 μl and then converted to pmol / ml medium (3 days production) by dividing these values by 100. Mean ± SEM of triplicate measurements of 5-6 wells for each condition. E. M.M. The value was expressed as Each experiment was verified at least twice using cells from different calvarial preparations.

Example 5: Effect of Trm and RLMP on in vitro mineralization In non-stimulated cell culture systems, there was little apparent effect on either total sense bone or nodules on either sense or antisense oligonucleotides. It was. However, when ROB was stimulated with Trm, antisense oligonucleotides to RLMP inhibited nodule mineralization> 95%. Addition of exogenous BMP-6 to oligonucleotide treated cultures did not rescue the mineralization of nodules treated with RLMP antisense.

  Osteocalcin has long been synonymous with bone mineralization, and osteocalcin levels have been correlated with nodule production and mineralization. RLMP antisense oligonucleotides significantly reduced osteocalcin production, but the number of nodules in cultures treated with antisense did not change significantly. In this case, addition of exogenous BMP-6 only rescued osteocalcin production in cultures treated with RLMP antisense by 10-15%. This suggested that the action of RLMP is downstream of BMP-6 and is more specific than BMP-6.

Example 6: Collection and purification of RNA Cellular RNA is collected from a duplicate well of ROB (prepared according to Examples 1 and 2 in a 6-well culture plate) using 4M guanidine isothiocyanate (GIT) solution. And got a triple for statistics. Briefly, culture supernatant was aspirated from the wells, and then the wells were overlaid with 0.6 ml of GIT solution per duplicate well collection. After adding the GIT solution, the plate was rotated for 5-10 seconds (as constant as possible). Samples were stored at -70 ° C for up to 7 days before further processing.

RNA was purified with slight modifications to the standard method according to Sambrook et al. Molecular Cloning: a Laboratory Manual, Chapter 7.19, 2nd Edition, Cold Spring Harbor Press (1989). Briefly, 60 μl of 2.0 M sodium acetate (pH 4.0), 550 μl of phenol (water saturated) and 150 μl of chloroform: isoamyl alcohol (49: 1) were added to the melted sample. After vortexing, the sample was centrifuged (10000 × g; 20 minutes; 4 ° C.), the aqueous phase was transferred to a new tube, 600 μl of isopropanol was added, and the RNA was allowed to precipitate at −20 ° C. overnight.

  After overnight incubation, the samples were centrifuged (10000 × g; 20 minutes) and the supernatant was gently aspirated. The pellet was resuspended in 400 μl DEPC-treated water, extracted once with phenol: chloroform (1: 1), extracted with chloroform: isoamyl alcohol (24: 1), and 40 μl sodium acetate (3.0 M; pH 5 .2) and 1.0 ml absolute ethanol were added followed by overnight precipitation at -20 ° C. To recover cellular RNA, the samples were centrifuged (10000 × g; 20 minutes), washed once with 70% ethanol, air-dried for 5-10 minutes, and resuspended in 20 μl DEPC-treated water. The RNA concentration was calculated from the optical density measured with a spectrophotometer.

Example 7: Reverse Transcription Polymerase Chain Reaction 25
Heated total RNA (5 μg in DEPC-H ₂ O in a total volume of 10.5 μl heated for 5 minutes at 65 ° C.), 4 μl 5 × MMLV-RT buffer, 2 μl dNTP, 2 μl dT17 primer (10 pmol / ml), 0 Added to tubes containing 5 μl RNAsin (40 U / ml) and 1 μl MMLV-RT (200 units / μl). Samples were incubated at 37 ° C. for 1 hour, followed by incubation at 95 ° C. for 5 minutes to inactivate MMLV-RT. Samples were diluted by adding 80 μl of water.

Using standard methodology, reverse transcribed samples (5 μl) were subjected to polymerase chain reaction (total volume 50 μl). Briefly, the sample is made of water and an appropriate amount of PCR buffer, 25 mM MgCl ₂ , dNTPs, glyceraldehyde 3-phosphate dehydrogenase (GAP, housekeeping gene) and / or BMP-6 forward and reverse primers, Added to tubes containing ³² P-dCTP and Taq polymerase. Unless stated otherwise, the primers were standardized for consistent reaction over 22 cycles (94 ° C., 30 ″; 58 ° C., 30 ″; 72 ° C., 20 ″).

Example 8: Quantification of RT-PCR product by polyacrylamide gel electrophoresis (PAGE) and phosphoimager analysis 5 μl / tube loaded dye is added to RT-PCR product, mixed and heated at 65 ° C. for 10 minutes And centrifuged. 10 μl of each reaction was subjected to PAGE (12% polyacrylamide: bis; 15 V / well; constant current) under standard conditions. The gel is then incubated for 30 minutes in gel storage buffer (10% v / v glycerol, 7% v / v acetic acid, 40% v / v methanol, 43% deionized water) and vacuum dried for 1-2 hours. (80 ° C.) and developed in an electrically enhanced phosphorescent imaging system for 6-24 hours. The visualized band was analyzed. The counts per band were plotted on a graph.

Example 9: Differential Display PCR
RNA was extracted from cells stimulated with glucocorticoid (Trm, 1 nM). Total RNA heated and treated with DNase (5 μg in DEPC-H ₂ O in a total volume of 10.5 μl heated for 5 minutes at 65 ° C.) as described in Example 7, but with MMLV-RT primer As a result, reverse transcription was performed using HT ₁₁ M (SEQ ID NO: 4). As described above, but generated using a variety of commercial primer sets (eg, HT ₁₁ G (SEQ ID NO: 4) and H-AP-10 (SEQ ID NO: 5); GeneHunter Corp, Nashville, TN) The cDNA was amplified by PCR. Radiolabeled PCR products were fractionated by gel electrophoresis on a DNA sequencing gel. After electrophoresis, the resulting gel was vacuum dried and exposed to an autoradiograph overnight. A band corresponding to the differentially expressed cDNA was excised from the gel, and Conner et al ., Proc. Natl. Acad. Sci. Re-amplification by PCR using the method of USA, 88, 278 (1983). The product of PCR reamplification was cloned into the vector PCR-11 (TA cloning kit; InVitrogen, Carlsbad, CA).

Example 10: Screening of UMR106 rat osteosarcoma cell cDNA library UMR106 library (2.5 × ^{10 10} pfu / ml) was plated on agar plates (LB bottom agar) at 5 × 10 ⁴ pfu / ml and the plate was incubated at 37 ° C. Incubated overnight. The filter membrane was placed on the plate for 2 minutes. Upon removal, the filter was denatured, rinsed, dried and UV cross-linked. The filters were then incubated for 2 hours at 42 ° C. in prehybridization buffer (2XPIPES [pH 6.5], 5% formamide, 1% SDS and 100 μg / ml denatured salmon sperm DNA). A 260 base pair radiolabeled probe (SEQ ID NO: 3; ³² P-labeled by random priming) was added to the entire hybridization mixture / filter and then hybridized at 42 ° C. for 18 hours. Membranes were washed once at room temperature (10 minutes, 1 × SSC, 0.1% SDS) and three times at 55 ° C. (15 minutes, 0.1 × SSC, 0.1% SDS).

  After washing, the membrane was analyzed by autoradiography as described above. Positive clones were plaque purified. The second filter was used for 4 minutes and the method was repeated to minimize false positives. Plaque purified clones were rescued as lambda SK (−) phagemids. The cloned DNA was sequenced as described below.

Example 11: Sequencing of clones The sequence of the cloned cDNA insert was determined by standard methods. Ausubel et al. , Current Protocols in Molecular Biology, Wiley Interscience (1988). Briefly, the appropriate concentrations of termination mixture, template and reaction mixture were subjected to the appropriate cycling protocol (95 ° C. for 30 seconds; 68 ° C. for 30 seconds; 72 ° C. for 60 seconds; x25). A stop mix was added to terminate the sequencing reaction. After heating at 92 ° C. for 3 minutes, the sample was loaded onto a denaturing 6% polyacrylamide sequencing gel (29: 1 acrylamide: bisacrylamide). Samples were electrophoresed at 60 volts and constant current for about 4 hours. After electrophoresis, the gel was vacuum dried and autoradiographed.

  The autoradiograph was analyzed manually. Generated using the BLASTN program with default parameters set against a database maintained by the National Center for Biotechnology Information (NIH, Bethesda, MD; http://www.ncbi.nlm.nih.gov/) The sequence was screened. Based on the sequence data, new sequencing primers were prepared and the process was repeated until the entire gene was sequenced. All sequences were confirmed at least 3 times in both directions.

  Nucleotide and amino acid sequences were also analyzed using the PCGENE software package (version 16.0). The percent homology value of nucleotide sequences was calculated by the program NALIGN using the following parameters: weight of non-matched nucleotides, 10; weight of non-match gaps, 10; maximum number of nucleotides to consider, 50; Minimum number of nucleotides, 50.

For amino acid sequences, percent homology values were calculated using PALIGN. A value of 10 was chosen for both open gap cost and unit gap cost.
Example 12: Cloning of RLMP cDNA The differential display PCR amplification product described in Example 9 contained a major band of approximately 260 base pairs. This sequence was used to screen a rat osteosarcoma (UMR106) cDNA library. Positive clones were subjected to nested primer analysis to obtain primer sequences necessary for amplifying full-length cDNA (SEQ ID NOs: 11, 12, 29, 30 and 31). One of the positive clones selected for further study was designated clone 10-4.

Sequence analysis of the full-length cDNA in clone 10-4, determined by nested primer analysis, shows that clone 10-4 contains the original 260 base pair fragment identified by differential display PCR. It was. Clone 10-4 (1696 base pairs; SEQ ID NO: 2) contains a 1371 base pair open reading frame encoding a protein having 457 amino acids (SEQ ID NO: 1). The termination codon TGA is present at nucleotides 1444-1446. A polyadenylation signal at nucleotides 1675-1680 and the adjacent poly (A) ⁺ tail were present in the 3 ′ non-coding region. Two potential N-glycosylation sites, Asn-Lys-Thr and Asn-Arg-Thr, were at amino acid positions 113-116 and 257-259 of SEQ ID NO: 1, respectively. Two potential cAMP and cGMP dependent protein kinase phosphorylation sites, Ser and Thr, were found at amino acid positions 191 and 349, respectively. There were five potential protein kinase C phosphorylation sites, Ser or Thr, at amino acid positions 3,115,166,219,442. One potential ATP / GTP binding site motif A (P-loop), Gly-Gly-Ser-Asn-Asn-Gly-Lys-Thr, was determined to be at amino acid positions 272-279.

  In addition, two highly conserved putative LIM domains were found at amino acid positions 341-391 and 400-451. The putative LIM domain in this newly identified rat cDNA clone showed considerable homology with the LIM domains of other known LIM proteins. However, the overall homology with other rat LIM proteins was less than 25%. RLMP (also referred to as 10-4) has 78.5% amino acid homology with human Enigma protein (see US Pat. No. 5,504,192), but the closest rat homologue, CLP- For 36 and RIT-18, they have only 24.5% and 22.7% amino acid homology, respectively.

Example 13: Northern blot analysis of RLMP expression 30 μg of ROB-derived total RNA prepared according to Examples 1 and 2 was size fractionated by formaldehyde gel electrophoresis in 1% agarose flatbed gel and nylon The membrane was transblotted osmotically. Blots were probed by random priming with a 600 base pair Eco RI fragment of full length 10-4 cDNA labeled with ³² P-dCTP.

  Northern blot analysis showed that a 1.7 kb mRNA species hybridizes with the RLMP probe. RLMP mRNA was upregulated approximately 3.7-fold in ROB 24 hours after exposure to BMP-6. ROB stimulated with BMP-2 or BMP-4 did not show upregulation of RMLP expression after 24 hours.

Example 14: Statistical Methods For each reported nodule / osteocalcin result, from a representative experiment, using data from 5-6 wells, the mean ± S. E. M.M. Was calculated. A graph is shown using data normalized to the maximum value of each parameter, and the number of nodules, mineralized area and osteocalcin can be graphed simultaneously.

  For each of the reported RT-PCR, RNase protection assay or Western blot analysis, using data from triplicate samples of representative experiments, the mean ± S. E. M.M. It was determined. The graph can be shown normalized to either day 0 or the negative control and expressed as a fold increase over the control value.

Statistical significance was assessed using one-way analysis of variance with Bonferroni's post hoc multiple comparison correction, as appropriate. D. V. Huntsberger , “The Analysis of Variance”, Elements of Statistical Variance, P.A. Billingsley (supervised), Allyn & Bacon Inc. Boston, Massachusetts, 298-330 (1977) and SigmaStat, Jandel Scientific, Corte Madera, California. The alpha level of significance was defined as p <0.05.

Example 15: Detection of rat LIM mineralized protein by Western blot analysis
England et al ., Biochim. Biophis. Acta, 623, 171 (1980) and Timer et al ., J. MoI. Biol. Chem. , 268, 24863 (1993), and polyclonal antibodies were prepared.

HeLa cells were transfected with pCMV2 / RLMP. Proteins were harvested from the transfected cells according to the method of Hair et al. , Leukemia Research, 20, 1 (1996). Towbin et al ., Proc. Natl. Acad. Sci. USA, 76: 4350 (1979), Western blot analysis of native RLMP was performed.

Example 16: Synthesis of a human PCR product derived from the rat LMP specific sequence (RLMPU) Based on the sequence of the rat LMP-1 cDNA, forward and reverse PCR primers (SEQ ID NOs: 15 and 16) were synthesized and unique. Of 223 base pairs was PCR amplified from rat LMP-1 cDNA. Similar PCR products were isolated from human MG63 osteosarcoma cell cDNA using the same PCR primers.

RNA was collected from MG63 osteosarcoma cells grown in T-75 flasks. The culture supernatant is removed by aspiration and the flask is overlaid with 3.0 ml of GIT solution per duplicate, swirled for 5-10 seconds, and the resulting solution is added to a 1.5 ml Eppendorf tube (0.6 ml / tube). 6 test tubes). RNA was purified with slight modifications of the standard method. See, for example, Sambrook et al. , Molecular Cloning: A Laboratory Manual, Chapter 7, page 19, Cold Spring Harbor Laboratory Press (1989) and Boden et al. , Endocrinology, 138, 2820-28. Briefly, 60 μl of 2.0 M sodium acetate (pH 4.0), 550 μl of water saturated phenol and 150 μl of chloroform: isoamyl alcohol (49: 1) were added to a 0.6 ml sample. After addition of these reagents, the samples were vortexed, centrifuged (10000 × g; 20 minutes; 4 ° C.), and the aqueous phase was transferred to a new tube. Isopropanol (600 μl) was added and RNA was precipitated overnight at −20 ° C. Samples were centrifuged (10000 × g; 20 minutes) and the supernatant was gently aspirated. The pellet was resuspended in 400 μl DEPC-treated water, extracted once with phenol: chloroform (1: 1), extracted with chloroform: isoamyl alcohol (24: 1), and 40 μl sodium acetate (3.0 M; pH 5 .2) and 1.0 ml absolute ethanol were precipitated overnight at -20 ° C. After precipitation, the samples were centrifuged (10000 × g; 20 minutes), washed once with 70% ethanol, air dried for 5-10 minutes, and resuspended in 20 μl DEPC-treated water. The RNA concentration was obtained from the optical density.

Total RNA (5 μg in a total volume of 10.5 μl DEPC-H ₂ O) was heated at 65 ° C. for 5 minutes and then 4 μl 5X MMLV-RT buffer, 2 μl dNTP, 2 μl dT17 primer (10 pmol / ml), Added to tubes containing 0.5 μl RNAsin (40 U / ml) and 1 μl MMLV-RT (200 units / μl). The reaction was incubated at 37 ° C. for 1 hour. Then, MMLV-RT was inactivated by heating at 95 ° C. for 5 minutes. Samples were diluted by adding 80 μl of water.

Using standard methodologies, transcribed samples (5 μl) were subjected to polymerase chain reaction (total volume 50 μl). Boden et al. , Endocrinology, 138, 2820-2828 (1997); Ausubel et al. , “Quantation of Rare DNAs by the Polymer Chain Reaction”, Current Protocols in M. 15, Current Protocols in MoI. ). Briefly, samples contain water and appropriate amounts of PCR buffer (25 mM MgCl ₂ , dNTP, forward and reverse primers (for RLMPU; SEQ ID NOs: 15 and 16), ³² P-dCTP, and DNA polymerase To detect radioactive bands consistently in 22 cycles, and to amplify PCR products for use as screening probes, consistently react in 33 cycles. Primers were designed (94 ° C. for 30 seconds; 58 ° C. for 30 seconds; 72 ° C. for 20 seconds).

  Sequencing of the MG63 osteosarcoma-derived PCR product purified on an agarose gel resulted in a sequence with greater than 95% homology to the RLMPU PCR product. This sequence is referred to as HLMMP specific region (HLMPU; SEQ ID NO: 6).

Example 17: Screening for reverse transcriptase-derived MG63 cDNA Screened by PCR using specific primers (SEQ ID NOs: 16 and 17) as described in Example 7. The 717 base pair MG63 PCR product was agarose gel purified and sequenced using predefined primers (SEQ ID NOs: 12, 15, 16, 17, 18, 27 and 28). The sequence was confirmed at least twice in both directions. The MG63 sequence was aligned with each other and then aligned with the full-length rat LMP cDNA sequence to yield a partial human LMP cDNA sequence (SEQ ID NO: 7).

Example 18: Screening of human heart cDNA library Based on Northern blot experiments, it was determined that LMP-1 is expressed at different levels by several different tissues, including human myocardium. Therefore, a human heart cDNA library was examined. The library was plated on agar plates (LB bottom agar) at 5 × 10 ⁴ pfu / ml and the plates were grown overnight at 37 ° C. The filter membrane was placed on the plate for 2 minutes. The filters are then denatured, rinsed, dried, UV cross-linked, and in prehybridization buffer (2XPIPES [pH 6.5]; 5% formamide, 1% SDS, 100 g / ml denatured salmon sperm DNA) And incubated at 42 ° C. for 2 hours. Radiolabeled LMP specific 223 base pair probe ( ³² P, random primer label; SEQ ID NO: 6) was added and allowed to hybridize at 42 ° C. for 18 hours. After hybridization, the membrane was washed once at room temperature (10 minutes, 1 × SSC, 0.1% SDS) and three times at 55 ° C. (15 minutes, 0.1 × SSC, 0.1% SDS). Double positive plaque-purified heart library clones were identified by autoradiography and rescued as lambda phagemids according to the manufacturer's protocol (Stratagene, La Jolla, CA).

  Restriction digests of positive clones produced cDNA inserts of various sizes. Inserts longer than 600 base pairs in length were selected for initial screening by sequencing. These inserts were sequenced by standard methods as described in Example 11.

  One clone, number 7, was also subjected to automated sequence analysis using primers corresponding to SEQ ID NOs: 11-14, 16 and 27. The sequences obtained by these methods were routinely 97-100% homologous. Clone 7 (partial human LMP-1 cDNA from cardiac library; SEQ ID NO: 8) contained a sequence having greater than 87% homology with the rat LMP cDNA sequence in the translation region.

Example 19: Determination of full length human LMP-1 cDNA Using the overlapping region of the MG63 human osteosarcoma cell cDNA sequence and the human heart cDNA clone 7 sequence, these two sequences were juxtaposed and a 1644 base pair full length human cDNA. The sequence was obtained. The two sequences were juxtaposed using the program NALIGN in the PCGENE software package. The overlapping region of the two sequences constitutes approximately 360 base pairs with complete homology except for the single nucleotide substitution at nucleotide 672 of the MG63 cDNA (SEQ ID NO: 7), and clone 7 has a “ It had “A” instead of “G” (SEQ ID NO: 8).

  Using the “G” substitution of the MG63 osteosarcoma cDNA clone, the two parallel sequences were ligated using SEQIN, another subprogram of PCGENE. The resulting sequence is shown in SEQ ID NO: 9. Parallelization of rat LMP-1 cDNA and novel human-derived sequences was achieved with NALIGN. The full-length human LMP-1 cDNA sequence (SEQ ID NO: 9) is 87.3% homologous to the translated portion of the rat LMP-1 cDNA sequence.

Example 20: Determination of the amino acid sequence of human LMP-1 The putative amino acid sequence of human LMP-1 was determined with the PCGENE subprogram TRANSL. The open reading frame in SEQ ID NO: 9 encodes a protein comprising 457 amino acids (SEQ ID NO: 10). Using the PCGENE subprogram Palign, the human LMP-1 amino acid sequence was found to be 94.1% homologous to the rat LMP-1 amino acid sequence.

Example 21: Determination of the 5 'untranslated region of human LMP cDNA MG63 5' cDNA was amplified by nested RT-PCR of MG63 total RNA using the 5 'rapid amplification of cDNA ends (5'RACE) protocol. This method involved first strand cDNA synthesis using a lock-dock oligo (dT) primer at two degenerate nucleotide positions at the 3 ′ end ( Chenchik et al. , CLONTECHniques, X: 5 (1995); Borson et al. , PC Methods Applic., 2, 144 (1993)). Second strand synthesis is performed using a cocktail of E. coli DNA polymerase 1, RNase H, and E. coli DNA ligase according to the method of Gubler et al. , Gene, 2,263 (1983). After generating a blunt end using T4 DNA polymerase, the double-stranded cDNA was ligated to the fragment (5′-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3 ′) (SEQ ID NO: 19). Prior to RACE, the adapter-ligated cDNA was diluted to a concentration (1:50) suitable for the Marathon RACE reaction. The double stranded cDNA with the adapter linked can then be used immediately for specific cloning.

  Using the adapter-specific oligonucleotide, 5′-CCATCCTAATACGACTCACTATAGGGC-3 ′ (AP1) (SEQ ID NO: 20) as the sense primer, and the gene-specific primer (GSP) from the unique region (HLMPU) described in Example 16 First cycle PCR was performed. Nested primers GSP1-HLMPU (antisense / reverse primer) (SEQ ID NO: 23) and GSP2-HLMPUF (SEQ ID NO: 24) (see Example 16; sense / forward primer) were used for the second cycle. PCR was performed. PCR was performed using a commercial kit (Advantage cDNA PCR Core Kit; ClonTech Laboratories Inc., Palo Alto, Calif.) That is antibody mediated but otherwise utilizes standard hot start protocols. PCR conditions for MG63 cDNA included initial hot start denaturation (94 ° C. for 60 seconds) followed by: 94 ° C. for 30 seconds; 60 ° C. for 30 seconds; 68 ° C. for 4 minutes; 30 cycles. The first cycle PCR product was approximately 750 base pairs in length, while the nested PCR product was approximately 230 base pairs. The first cycle PCR product was cloned into a linearized pCR2.1 vector (3.9 Kb). The insert was sequenced in both directions using M13 forward and reverse primers (SEQ ID NO: 11; SEQ ID NO: 12).

Example 22: Determination of full length human LMP-1 cDNA with 5 'prime UTR Overlapping MG63 human osteosarcoma cell cDNA 5'-UTR sequence (SEQ ID NO: 21), MG63 717 base pair sequence (Example 17; SEQ ID NO: 8 ) And the human heart cDNA clone 7 sequence (Example 18) were juxtaposed to yield a novel human cDNA sequence (SEQ ID NO: 22) of 1704 base pairs. Parallelism was achieved using NALIGN (both PCGENE and Omiga 1.0; Intelligenetics). The overlapping sequence comprised almost all 717 base pair regions (Example 17) and was 100% homologous. Parallel sequence ligation was achieved with SEQIN.

Example 23: Construction of LIM protein expression vector A pHIS-5ATG LMP-1s expression vector was constructed using the sequences described in Example 17 and Example 18. A 717 base pair clone (Example 17; SEQ ID NO: 7) was digested with Cla I and Eco RV. A small fragment (˜250 base pairs) was gel purified. Clone 7 (Example 18; SEQ ID NO: 8) was digested with Cla I and Xba I and the 1400 base pair fragment was gel purified. The isolated 250 and 1400 base pair restriction fragments were ligated to form a ˜1650 base pair fragment.

  A single nucleotide substitution in clone 7 (compared to the 717 base pair PCR sequence and the original rat sequence) resulted in a stop codon at translated base pair 672. Because of this stop codon, a partially excised (short) protein referred to hereinafter as LMP-1s was encoded. This was the construct used in the expression vector (SEQ ID NO: 32). SEQ ID NO: 32 and 5'RACE sequence (SEQ ID NO: 21) were juxtaposed to generate a full length cDNA sequence (SEQ ID NO: 33) containing a 5'UTR. Subsequently, the amino acid sequence of LMP-1s (SEQ ID NO: 34) was deduced to be a 223 amino acid protein, and when confirmed by Western blot (as in Example 15), the expected molecular weight was ˜23.7 kD. Electrophoresed.

The pHis-ATG vector (InVitrogen, Carlsbad, CA) was digested with Eco RV and Xba I. The vector was recovered and then a 650 base pair restriction fragment was ligated to the linearized pHis-ATG. The ligated product was cloned and amplified. The pHis-ATG-LMP-1s expression vector, also referred to as pHIS-A, containing the insert HLMP-1s was purified by standard methods.

Example 24: In vitro bone nodule formation and mineralization-induced rat calvarial cells using LMP expression vectors were isolated and grown in the secondary culture described in Example 1. Cultures were not stimulated or stimulated with glucocorticoid (GC) as described in Example 1. Secondary rat calvarial osteoblast cultures were transfected with 3 μg / well of each vector according to Example 25 using a modification of the Superfect reagent (Qiagen, Valencia, Calif.) Transfection protocol.

  Mineralized nodules were visualized by von Kossa staining as described in Example 3. Human LMP-1s gene product overexpression alone induced bone nodule formation (˜203 nodules / well) in vitro. The nodule level was approximately 50% of that induced in the GC positive control (˜412 nodules / well). Other positive controls include pHisA-LMP-rat expression vector (˜152 nodes / well) and pCMV2 / LMP-rat-Fwd expression vector (˜206 nodes / well), while negative controls include pCMV2 / LMP-rat-Rev expression vector (~ 2 nodes / well) and untreated (NT) plates (~ 4 nodes / well) were included. These data demonstrate that the human cDNA was at least as osteoinductive as the rat cDNA. The effect is lower than that observed with GC stimulation, most likely because the expression vector was at a suboptimal dose.

Example 25: Cell differentiation induced by in vitro and in vivo LMP
Rat LMP cDNA in clone 10-4 (see Example 12) was excised from the vector by double digesting the clone with Not I and Apa I overnight at 37 ° C. The vector pCMV2 MCS (InVitrogen, Carlsbad, CA) was digested with the same restriction enzymes. Both clone 10-4 and the linear cDNA fragment from pCMV2 were gel purified, extracted and ligated using T4 ligase. The ligated DNA was gel purified, extracted, and used to transform E. coli JM109 cells for amplification. Positive agar colonies were picked, digested with Not I and Apa I, and restriction digests were examined by gel electrophoresis. Stock cultures of positive clones were prepared.

A reverse vector was prepared in a similar manner, except that the restriction enzymes used were Xba I and Hind III. Since these restriction enzymes were used, the LMP cDNA fragment derived from clone 10-4 was inserted into pRc / CMV2 in the reverse direction (ie, untranslatable). The produced recombinant vector is called pCMV2 / RLMP.

  An appropriate volume of pCMV10-4 (60 nM final concentration is optimal [3 μg]; for this experiment, a final concentration in the range of 0-600 nM / well [0-30 μg / well] is preferred) with minimal Eagle medium (MEM ) To a final volume of 450 μl and vortexed for 10 seconds. Superfect was added (7.5 μl / ml final solution), the solution was vortexed for 10 seconds and then incubated at room temperature for 10 minutes. After this incubation, MEM supplemented with 10% FBS (1 ml / well; 6 ml / plate) was added and mixed by pipetting.

The resulting solution was then immediately pipetted onto the washed ROB culture (1 ml / well). Cultures were incubated for 2 hours at 37 ° C. in a humidified atmosphere containing 5% CO ₂ . The cells were then gently washed once with sterile PBS and the appropriate normal incubation medium was added.

  The results showed significant bone nodule formation in all rat cell cultures induced with pCMV10-4. For example, cells transfected with pCMV10-4 gave 429 nodules / well. Positive control cultures exposed to Trm yielded 460 nodules / well. In contrast, a negative control that received no treatment yielded 1 nodule / well. Similarly, no nodules were observed when the cultures were transfected with pCMV10-4 (reverse direction).

To demonstrate de novo bone formation in vivo, bone marrow was aspirated from the hind limbs of 4-5 week old normal rats (rnu / +; heterozygous for recessive athymic state). RBCs were lysed by washing the aspirated bone marrow cells in alpha MEM, centrifuging, and resuspending the pellet in 0.83% NH ₄ Cl in 10 mM Tris (pH 7.4). The remaining bone marrow cells were washed 3 times with MEM and transfected with 9 μg pCMV-LMP-1s (forward or reverse) per 3 × 10 ⁶ cells for 2 hours. The transfected cells were then washed twice with MEM and resuspended at a concentration of 3 × 10 ⁷ cells / ml.

  With a sterile pipette, the cell suspension (100 μl) was applied to a sterile 2 × 5 mm type I bovine collagen disc (Sulzer Orthopaedics, Wheatridge, CO). The disc was surgically implanted subcutaneously into the skull, chest, abdomen or spinal column of 4-5 week old athymic rats (rnu / rnu). From week 3 to week 4 the animals were sacrificed, at which time the disc or surgical area was excised and fixed in 70% ethanol. Fixed specimens were analyzed by radiography and undecalcified histological examinations were performed on 5 μm thick sections stained with Goldner's trichromatic stain. Experiments were also performed using a demineralized bone matrix (Osteeotech, Shrewsbury, NJ), instead of collagenized disks (devitalized).

  Radiography revealed a high level of mineralized bone formation, consistent with the original collagen disc shape containing LMP-1s transfected bone marrow cells. In the negative control (cells transfected with the reverse version of the LMP-1s cDNA, which does not encode a protein to be translated), no mineralized bone formation is formed and carrier absorption appears to be proceeding considerably. there were.

  Histology revealed new bone trabeculars lined with osteoblasts in the LMP-1s transfection implant. In the negative control, no bone was seen along the partial absorption of the carrier.

  Radiographs of further experiments where 18 sets of implants (9 negative controls pCMV-LMP-REV and 9 experimental pCMV-LMP-1s) were added alternately at the site between the lumbar and thoracic vertebrae of athymic rats Demonstrated that a 0/9 negative control implant showed bone formation between the vertebrae (vertebral fixation). All nine of the pCMV-LMP-1s treated implants showed solid bone fixation between the vertebrae.

Example 26: Synthesis of a pHIS-5'ATG LMP-1s expression vector 717 base pair clone (Example 17) from the sequences shown in Example 2 and Example 3 was cloned into Cla I and Eco RV (New England Biologicals, Massachusetts). Digested in state city. A small fragment (˜250 base pairs) was gel purified. Clone number 7 (Example 18) was digested with Cla I and Xba I. A 1400 base pair fragment was gel purified from the digest. The isolated 250 and 1400 base pair cDNA fragments were ligated by standard methods to form a ˜1650 bp fragment. The pHis-A vector (InVitrogen) was digested with Eco RV and Xba I. The linearized vector was recovered and ligated to the chimeric 1650 base pair cDNA fragment. The pHis-A-5′ATG LMP-1s expression vector, which is also cloned and amplified by standard methods and also referred to as the vector pHisA containing the insert HLMP-1s, has been deposited with the ATCC as described above. did.

Example 27: Induction of bone nodule formation and mineralization in vitro using pHis-5'ATG LMP-1s expression vector According to Example 1, rat calvarial cells were isolated and in secondary culture Allowed to grow. According to Example 1, the culture was not stimulated or stimulated with glucocorticoid (GC). Cultures were transfected with 3 μg of recombinant pHis-A vector DNA / well as described in Example 25. Mineralized nodules were visualized by von Kossa staining as described in Example 3.

  Only overexpression of the human LMP-1s gene product (ie, no GC stimulation) induced significant bone nodule formation (˜203 nodules / well) in vitro. This is approximately 50% of the amount of nodules (˜412 nodules / well) that result in a positive control of cells exposed to GC. Similar results were obtained in cultures transfected with pHisA-LMP-rat expression vector (˜152 nodes / well) and pCMV2 / LMP-rat-Fwd (˜206 nodes / well). In contrast, the negative control pCMV2 / LMP-Rat-Rev produced ˜2 nodules / well, while approximately 4 nodules / well were seen in the untreated plates. These data indicate that human LMP-1 cDNA was at least as osteoinductive as rat LMP-1 cDNA in this model system. The effects of this experiment were inferior to those observed with GC stimulation; in some, the effects were comparable.

Example 28: LMP induces secretion of soluble osteoinductive factor As described in Example 24, overexpression of RLMP-1 or HLMP-1s in rat calvarial osteoblast cultures resulted in a negative control Significantly more nodule formation occurred than was observed. To study the mechanism of action of LIM mineralized proteins, conditioned medium is collected at different time points, concentrated 10-fold, sterile filtered, and diluted to the original concentration in medium containing fresh serum. And applied to untransfected cells for 4 days.

  On day 4, conditioned medium collected from cells transfected with RLMP-1 or HLMP-1s was as effective as direct overexpression of RLMP-1 in the transfected cells in inducing nodule formation. Conditioned media from cells transfected with reverse RLMP-1 or HLMP-1 had no apparent effect on nodule formation. Also, conditioned media collected from LMP-1 transfection cultures prior to day 4 did not induce nodule formation. These data suggest that LMP-1 expression caused the synthesis and / or secretion of a soluble factor that was not found in effective amounts in the medium until 4 days after transfection.

  As a result of the overexpression of rLMP-1, osteoinductive factors were secreted into the medium, so Western blot analysis was used to determine whether LMP-1 protein was present in the medium. By conventional means, an antibody specific for LMP-1 (QDPDEE) was used to assess and detect the presence of RLMP-1 protein. LMP-1 protein was found only in the cell layer of the culture and was not detected in the medium.

  Partial purification of osteoinductive soluble factors was achieved by standard 25% and 100% ammonium sulfate cuts followed by DE-52 anion exchange batch chromatography (100 mM or 500 mM NaCl). All activity was seen with high ammonium sulfate, high NaCl fractions. Such localization was consistent with the presence of a single factor involved in media acclimation.

Example 29: Low-dose adenovirus-mediated gene therapy in lumbar fusion This study shows that in normal, immunocompetent rabbits, an optimal dose of LMP-1 cDNA (SEQ ID NO: 2) Adenovirus delivery was determined.

A replication-deficient human recombinant adenovirus was constructed using LMP-1 cDNA (SEQ ID NO: 2) driven by the CMV promoter using the Adeno-Quest ^™ kit (Quantum Biotechnologies, Inc., Montreal). A commercially available (Quantum Biotechnologies, Inc., Montreal), recombinant adenovirus containing the beta-galactosidase gene was used as a control.

First, an in vitro dose response experiment was performed and transduced at a multiplicity of viral infection (“MOI”) of 0.025, 0.25, 2.5, or 25 plaque forming units (pfu) per cell for 60 minutes. The optimal concentration of adenovirus carrying LMP-1 (“AdV-LMP-1”) to induce bone differentiation in rat calvarial osteoblast cultures was determined. Positive control cultures were differentiated by exposure to 10 ⁹ M glucocorticoid (“GC”) for 7 days. Negative control cultures were left untreated. On day 14, after von Kossa staining of the culture, the number of mineralized bone nodules was counted, and the osteocalcin level (pmol / ml) secreted into the medium was measured by radioimmunoassay (mean ± SEM).

  The experimental results are shown in Table 1. Essentially no spontaneous nodules were formed in the untreated negative control culture. This data shows that an MOI equal to 0.25 pfu / cell is most effective in osteoinducing bone nodules and achieves a level comparable to the positive control (GC). Lower and higher doses of adenovirus were less effective.

Table 1

  Thereafter, in vivo experiments were performed to determine whether the optimal dose in vitro could promote intercostal spinal fixation in skeletal mature New Zealand white rabbits. Nine rabbits were anesthetized and 3 cc of bone marrow was aspirated from the distal thigh through the intercondylar fossa using an 18 gauge needle. The buffy coat was then isolated, transduced with AdV-LMP-1 for 10 minutes, and the cells were returned to the operating room for transplantation. Contains 8 million to 15 million self-nucleated buffy coat cells exfoliated and transduced with either AdV-LMP-1 (MOI = 0.4) or AdV-BGal (MOI = 0.4) The carrier (either deactivated rabbit bone matrix or collagen sponge) was inserted and a single level of posterior lumbar arthrodesis was performed. After 5 weeks, the rabbits were euthanized and spinal fixation was assessed by hand palpation, planar x-ray, CT scan, and non-decalcified histology.

  The spinal fixation site administered AdV-LMP-1 induced a continuous solid spinal fixation mass in all nine rabbits. In contrast, sites administered AdV-BGal, or lower doses of AdV-LMP-1 (MOI = 0.04) formed little or no bone and were comparable to carrier alone (<40%). Resulting in a rate of spinal fixation. These results were consistent with those assessed by hand palpation, CT scan, and histology. However, planar radiography sometimes overestimated the amount of bone present, especially at the control site. LMP-1 cDNA delivery and osteoinduction were successful with both carrier materials tested. There was no evidence of systemic or local immune responses to adenoviral vectors.

  These results show very difficult and consistent bone guidance in the previously validated rabbit spinal fixation model. In addition, protocols using autologous bone marrow cells ex vivo gene transduced (10 minutes) during surgery, rather than other methods that require overnight transduction or cell growth in culture for weeks. More clinically feasible. Furthermore, the most effective dose of recombinant adenovirus (MOI = 0.25) was substantially lower than the dose reported in other gene therapy applications (MOI 40-500). This is thought to be because LMP-1 is an intracellular signaling molecule and may have a strong signal amplification cascade. In addition, when changing from cell culture to animal experiments, other growth factors usually require dose escalation, so AdV-LMP- at the same concentration that induces bone in cell culture. The observation that 1 was effective in vivo was also surprising. Taken together, these observations allow local gene therapy to deliver LMP-1 cDNA using adenovirus and minimize the negative impact of immune responses against adenoviral vectors due to the low dose required. It is shown that it could be limited.

Example 30: Use of Peripheral Venous Blood Nucleated Cells (Buffy Coat) for Gene Therapy Generating Bone with LMP-1 cDNA In 4 rabbits, the transduced cells are not bone marrow but venous blood A spinal fusion was performed as described above (Example 29) except that the buffy coat was derived from. These cells were transfected with Adeno-LMP or pHIS-LMP plasmids and the results were as successful as when using bone marrow cells. This finding makes clinical use of gene therapy more clinical because using normal venous blood cells for gene delivery avoids painful bone marrow collection under general anesthesia and yields starting material with twice as many cells per ml. Make it feasible.

Example 31: Isolation of human LMP-1 splicing variants Intron / exon mRNA transcriptional splicing variants are relatively common regulatory mechanisms of signal transduction and cell / tissue development. Various gene splicing variants have been shown to alter protein-protein, protein-DNA, protein-RNA, and protein-substrate interactions. Splicing variants can also regulate the tissue specificity of gene expression, allowing different types (and thus functions) to be expressed in diverse tissues. Splicing variants are common regulatory events in cells. It is possible that LMP splicing variants can cause effects in other tissues, such as nerve regeneration, muscle regeneration, or other tissue development.

  To screen a human heart cDNA library for splicing variants of the HLMP-1 sequence, a PCR primer pair corresponding to the portion of SEQ ID NO: 22 was prepared. The forward PCR primer synthesized using standard techniques corresponds to nucleotides 35-54 of SEQ ID NO: 22. The primer has the following sequence:

  The reverse PCR primer, which is the reverse complement of nucleotides 820-839 of SEQ ID NO: 22, has the following sequence:

  Using a cycling protocol in which standard techniques are repeated 30 times at 94 ° C. for 30 seconds, 64 ° C. for 30 seconds, and 72 ° C. for 1 minute, and then incubated at 72 ° C. for 10 minutes, using forward and reverse PCR primers Thus, human heart cDNA (ClonTech, catalog number 7404-1) was screened for sequences similar to HLMP-1. The amplified cDNA sequence was gel purified and submitted to the Emory DNA Sequence Core Facility for sequencing. Clones were sequenced using standard techniques and sequenced with PCGENE (intelligentics; programs SEQUIN and NALIGN) to determine homology to SEQ ID NO: 22. Subsequently, two homologous nucleotide sequences with putative alternative splicing sites compared to SEQ ID NO: 22 were translated into their respective protein products with the Intelligenetic program TRANSL.

  One of these two novel human cDNA sequences (SEQ ID NO: 37) comprises 1456 bp:

  A reading frame shift caused by deletion of the 119 bp fragment (between X) and addition of the 17 bp fragment (underlined) results in a partially excised gene product with the following derived amino acid sequence (SEQ ID NO: 38):

  This 423 amino acid protein shows 100% homology with the protein shown in SEQ ID NO: 10, except for the highlighted region (amino acids 94-99), which differs due to the nucleotide changes shown above.

  A second novel human heart cDNA sequence (SEQ ID NO: 39) comprises 1575 bp:

A reading frame shift caused by the addition of a 17 bp fragment (bold, italic, underlined) results in an early translation stop codon at positions 565-567 (underlined).
The derived amino acid sequence (SEQ ID NO: 40) consists of 153 amino acids:

  This protein shows 100% homology to SEQ ID NO: 10 up to amino acid 94, where a 17 bp fragment shown in the nucleotide sequence is added, resulting in a frameshift. Over amino acids 94-153, this protein is not homologous to SEQ ID NO: 10. Amino acids 154 to 457 of SEQ ID NO: 10 are not present due to the early stop codon shown in the nucleotide sequence.

Example 32: Genomic HLMP-1 Nucleotide Sequence Applicants have identified a genomic DNA sequence encoding HLMP-1, including putative regulatory elements associated with HLMP-1 expression. The entire genome sequence is shown in SEQ ID NO: 41. This sequence was derived from AC023788 (clone RP11-564G9), University of Washington Medical School Genome Sequencing Center, St. Louis, MO.

  The putative promoter region of HLMP-1 spans nucleotides 2,660 to 8,733 of SEQ ID NO: 41. This region includes, among other things, at least 10 potential glucocorticoid response elements (“GRE”) (nucleotides 6148-6153, 6226-6231, 6247-6252, 6336-6341, 6510-6515, 6552-6557, 6727-6732. , 6752-6757, 7738-7743, and 8255-8260), and 12 potential binding sequence sites (nucleotides) of Mothers' Sma-2 homologue ("SMAD") to Drosophila decapentaplegic. 3569-3575, 4552-4558, 4582-4588, 5226-5232, 6228-6234, 6649-6655, 6725-6731, 6930-6936, 73 9～7384,7738～7742,8073～8079, and 8378-8384), and comprises three TATA boxes (nucleotides 5910～5913,6932～6935, and 7380 to 7383). Eight of the three TATA boxes, all GREs, and SMAD binding sequences (“SBE”) are clustered in the region spanning nucleotides 5,841-8,733 of SEQ ID NO: 41. These control elements can be used, for example, to control the expression of exogenous nucleotide sequences that encode proteins involved in the osteogenic process. This would allow systemic administration of factors or genes involved in tissue differentiation and development, along with therapeutic factors or genes related to bone formation and bone repair.

  In addition to putative regulatory elements, 13 exons corresponding to the nucleotide sequence encoding HLMP-1 have been identified. These exons span the following nucleotides in SEQ ID NO: 41:

HLMP-2 has another exon (exon 5A), which spans nucleotides 14887-14904.
Example 33: Expression of HLMP-1 in intervertebral disc cells LIM mineralized protein-1 (LMP-1) is an intracellular protein capable of directing cell differentiation in bone and non-bone tissue. This example demonstrates that expression of human LMP-1 ("HLMP-1") in intervertebral disc cells increases proteoglycan synthesis and promotes a more chondrogenic phenotype. Furthermore, the effect of HLMP-1 expression on cellular gene expression was demonstrated by measuring aggrecan and BMP-2 gene expression. Lumbar disc cells were harvested from Sprague-Dawley rats by gentle digestion with enzymes and cultured in monolayers in DMEM / F12 supplemented with 10% FBS. These cells were then split into 6-well plates at approximately 200,000 cells per well and cultured for approximately 6 days until the cells reached approximately 300,000 cells per well. The medium was changed to 1% FBS DMEM / F12 and this was considered day 0.

  A replication deficient type 5 adenovirus comprising an HLMP-1 cDNA operably linked to a cytomegalovirus (“CMV”) promoter has been previously described, for example, in US Pat. No. 6,300,127. ing. The negative control adenovirus was identical except that the HLMP-1 cDNA was replaced with LacZ cDNA. For positive controls, uninfected cultures were incubated in the presence of BMP-2 continuously at a concentration of 100 nanograms / milliliter.

  On day 0, the culture was infected with adenovirus in 300 microliters of medium containing 1% FBS at 37 ° C. for 30 minutes. Optimal dose range for transgene expression by analyzing cells activated with adenovirus (“AdGFP”) containing the green fluorescent protein (“GFP”) gene by fluorescence activated cell sorting (“FACS”) It was determined. Adenovirus containing human LMP-1 cDNA (AdHLMP-1) (MOI of 0, 100, 300, 1000, or 3000), or adenovirus containing a LacZ marker gene (AdLacZ, 1000 MOI) (negative control) Cells were treated with. The medium was changed 3 and 6 days after infection.

  Proteoglycan production is measured by measuring sulfated glycosaminoglycan (sGAG) present in the medium (Day 0, Day 3 and Day 6) using a dimethylmethylene blue (“DMMB”) calorimetric assay. Estimated.

  To quantify aggrecan and BMP-2 mRNA, cells were harvested on day 6 and mRNA was extracted by the Trizol technique. MRNA was converted to cDNA using reverse transcriptase and used for real-time PCR to allow determination of the relative abundance of aggrecan and BMP-2 messages. In previous experiments, real-time primers for aggrecan and BMP-2 were designed and tested. Cybergreen technology was used. MRNA abundance was quantified using a standardized curve.

  For transfected cells, the cell morphology was documented using a light microscope. Cells became more rounded with AdHLMP-1 (MOI 1000) treatment, but not with AdLacZ treatment. AdLacZ infection did not significantly change cell morphology.

FACS analysis of rat disc cells infected with ADGFP at a MOI of 1000 showed that the highest proportion of cells (45%) were infected.
There was a dose-dependent increase between sGAG production and AdhLMP-1 MOI. These data are shown in FIG. 1, which shows sGAG production after overexpression of HLMP-1 at different MOIs in rat intervertebral disc cells. The results were normalized to day 0 untreated cells. Error bars correspond to the standard error of the mean. As shown in FIG. 1, sGAG production observed on day 3 was relatively small, indicating that there was a time lag between transfection and cellular production of GAG. Treatment with AdLacZ did not significantly change sGAG production. As also shown in FIG. 1, the optimal dose of AdhLMP-1 was 1000 MOI, resulting in a 260% increase in sGAG production over day 6 untreated controls. Higher or lower doses of AdhLMP-1 lead to a reduced response.

  The effect of AdhLMP-1 dosage (MOI) on sGAG production is further illustrated in FIG. FIG. 2 is a graph showing rat intervertebral disc sGAG levels after 6 days of treatment with different MOI AdhLMP-1. As can be seen from FIG. 2, the optimal dose of AdhLMP-1 was 1000 MOI.

  Aggrecan mRNA and BMP-2 mRNA production is shown in FIG. This figure demonstrates the increase in aggrecan and BMP-2 mRNA after HLMP-1 overexpression. On day 6, real-time PCR of mRNA extracted from rat intervertebral disc cells was performed to compare cells treated with 250 MOI of ADhLMP-1 and untreated ("NT") cells. The data in FIG. 3 is shown as a percent increase over the untreated sample. As illustrated in FIG. 3, a significant increase in aggrecan mRNA and BMP-2 mRNA was noted after AdhLMP-1 treatment. Increased BMP-2 expression suggests that BMP-2 is a downstream gene that mediates HLMP-1 stimulation of proteoglycan synthesis.

  These data demonstrate that transfection with AdhLMP-1 is effective in increasing proteoglycan synthesis in intervertebral disc cells. The viral dose leading to the highest transgene expression (MOI 1000) also led to the highest induction of sGAG, suggesting that there is a correlation between HLMP-1 expression and sGAG induction. These data indicate that HLMP-1 gene therapy is a method of increasing proteoglycan synthesis in the intervertebral disc and that HLMP-1 is an agent for treating intervertebral disc disease.

  FIG. 4A is a graph showing HLMP-1 mRNA expression 12 hours after infection with Ad-hLMP-1 of different MOI. In FIG. 4A, exogenous LMP-1 expression was induced with different doses (MOI) of Ad-hLMP-1 virus and quantified by real-time PCR. For the purpose of comparing this data, it is normalized to the HLMP-1 mRNA level of Ad-LMP-1 MOI 5. HLMP-1 was not detected in the negative control group without treatment (“NT”) or with Ad-LacZ treatment (“LacZ”). HLMP-1 mRNA levels reached an approximately 8-fold plateau at a MOI of 25 and 50 in a dose dependent manner.

  FIG. 4B is a graph showing sGAG production in the medium 3 to 6 days after infection. DMMB assay was used to quantify total sGAG production between 3 and 6 days after infection. The data of FIG. 4B is normalized to a control (ie no treatment) group. As can be seen from FIG. 4B, there was a dose-dependent increase in sGAG, and peaks of approximately 3-fold increase over control were achieved at MOIs of 25 and 50. In the negative control, 25 MOI Ad-LacZ did not lead to any increase in sGAG. In FIG. 4B, the results are expressed as the mean and SD of three samples.

FIG. 5 is a graph showing changes over time in sGAG production. As can be seen from FIG. 5, on day 3, sGAG production was significantly increased at MOIs of 25 and 50. On day 6, there was a dose-dependent increase in sGAG production in response to AdLMP-1. A plateau level of sGAG increase was achieved at an MOI of 25. As can also be seen from FIG. 5, treatment with AdLacZ (“LacZ”) did not significantly change sGAG production. For 6-9 samples, each result is expressed as mean and SD. In FIG. 5, “ ^** ” indicates a data point with a P value <0.01 relative to an untreated control.

FIGS. 6A and 6B are graphs showing the gene response to LMP-1 overexpression in rat annulus cells for aggrecan and BMP-2, respectively. Quantitative real-time PCR was performed 3 days after infection with 25 MOIs of Ad-LMP-1 (“LMP-1”). As can be seen from FIGS. 6A and 6B, aggrecan and BMP-2 gene expression was significantly increased after infection with Ad-LMP-1 compared to the untreated control (“NT”). Furthermore, treatment with 25 MOIs of AdLacZ (“LacZ”) did not significantly alter either aggrecan or BMP-2 gene expression compared to untreated controls. In FIGS. 6A and 6B, each result is expressed as mean and SD for 6 samples. 6A and 6B, “ ^** ” indicates a data point having a P value of P <0.01.

  FIG. 7 is a graph showing the time course of HLMP-1 mRNA levels in rat annulus cells after infection with AdLMP-1 at a MOI of 25. The data is expressed as a fold increase over 5 MOIs of AdLMP-1 after normalization using the replication factor of 18S and overexpressed LMP-1 primers. As can be seen from FIG. 7, HLMP-1 mRNA was significantly upregulated as early as 12 hours after infection. Furthermore, there was a significant increase in expression levels between day 1 and day 3. Each result in FIG. 7 is expressed as the average and SD of 6 samples.

FIG. 8 is a graph showing changes in mRNA levels of BMP and aggrecan in response to HLMP-1 overexpression. At various time points after infection with 25 MOI of Ad-hLMP-1, mRNA levels of BMP-2, BMP-4, BMP-6, BMP-7, and aggrecan were measured by real-time PCR. As can be seen from FIG. 8, BMP-2 mRNA was significantly up-regulated as early as 12 hours after infection with AdLMP-1. On the other hand, aggrecan mRNA was not up-regulated until 3 days after infection. Each result is expressed as the mean and SD of 6 samples. In FIG. 8, “ ^** ” indicates a data point with a P value <0.01 for AdLMP-1 infection relative to an untreated control.

  FIG. 9 is a graph showing the time course of sGAG production enhancement in response to HLMP-1 expression. For the data in FIG. 9, rat ring cells were infected with Ad-hLMP-1 at a MOI of 25. The medium was changed every 3 days after infection and assayed for sGAG in the DMMB assay. This data shows that sGAG production reaches a plateau at day 6 and is substantially maintained at day 9.

  FIG. 10 is a graph showing the effect of noggin (BMP antagonist) on sGAG production increase mediated by LMP-1. As seen in FIG. 10, infection of rat ring cells with 25 MOI of Ad-LMP-1 led to a 3-fold increase in sGAG produced between days 3-6. This increase was blocked by adding noggin (BMP antagonist) at concentrations of 3200 ng / ml and 800 ng / ml. However, as shown in FIG. 10, noggin did not significantly alter sGAG production in uninfected cells. As can also be seen in FIG. 10, stimulation with 100 ng / ml rhBMP-2 led to a 3-fold increase in sGAG production between 3 and 6 days after addition of BMP-2. 800 ng / ml Noggin also blocked this increase.

  FIG. 11 is a graph showing the effect of LMP-1 on sGAG in the medium after 6 days of monolayer culture. Data points are expressed as a fold increase over untreated cells. As shown in FIG. 11, delivery of LMP-1 and CMV promoter by an AAV vector is also effective in stimulating glycosaminoglycan synthesis by monolayer rat disc cells.

Table 2: SYBR Green RT-PCR and real-time PCR primer sequences

GAPDH in Table 2 indicates glyceraldehyde phosphate dehydrogenase.
Table 3: TaqMan® real-time PCR primer and probe sequences

  TaqMan® ribosomal RNA modulatory reagent (part number 4308329, Applied Biosystems, Foster City, Calif., USA) was used for the forward primer, reverse primer, and probe of the 18S ribosomal RNA (rRNA) gene.

All cited publications and patents are fully incorporated herein by reference.
The foregoing specification describes the principles of the present invention and includes examples provided for purposes of illustration, but upon reading this disclosure by those skilled in the art, it will be understood in form and detail without departing from the true scope of the invention. It will be recognized that various changes can be made.

The present invention may be better understood with reference to the accompanying figures.
FIG. 1 is a graph showing the production of sulfated glycosaminoglycan (sGAG) following expression of HLMP-1 by rat disc cells transfected with different MOIs. FIG. 2 is a graph showing the dose response of rat disc cells 6 days after infection with different MOI of AdHLMP-1. FIG. 3 is a graph showing the expression of aggrecan mRNA and BMP-2 mRNA by rat disc cells transfected with AdHLMP-1 6 days after transfection with a MOI of 250 virions / cell. FIG. 4A is a graph showing HLMP-1 mRNA expression 12 hours after infection with Ad-hLMP-1 of different MOI. FIG. 4B is a graph showing sGAG production in the medium 3 to 6 days after infection. FIG. 5 is a graph showing changes over time in sGAG production. FIG. 6A is a graph showing the gene response to LMP-1 overexpression in rat annulus cells for aggrecan. FIG. 6B is a graph showing the gene response to LMP-1 overexpression in rat annulus cells for BMP-2. FIG. 7 is a graph showing the time course of HLMP-1 mRNA levels in rat annulus cells after infection with AdLMP-1 at a MOI of 25. FIG. 8 is a graph showing changes in mRNA levels of BMP and aggrecan in response to HLMP-1 overexpression. FIG. 9 is a graph showing the time course of sGAG production enhancement in response to HLMP-1 expression. FIG. 10 is a graph showing that the increase in sGAG production mediated by LMP-1 is blocked by noggin. FIG. 11 is a graph showing the effect of LMP-1 on sGAG in the medium after 6 days of monolayer culture.

[Sequence Listing]

Claims (50)

A method for expressing a LIM mineralized protein in non-osseous mammalian cells comprising:
Said method comprising transfecting the cell with an isolated nucleic acid comprising a nucleotide sequence encoding a LIM mineralized protein linked to a promoter so as to be functional.   2. The method of claim 1, wherein the cell is capable of producing proteoglycan and / or collagen, and expression of the LIM mineralized protein stimulates proteoglycan and / or collagen synthesis in the cell. Isolated nucleic acid:
Hybridizes to nucleic acid molecules complementary to the full length of SEQ ID NO: 25 under standard conditions; and / or hybridizes to nucleic acid molecules complementary to the full length of SEQ ID NO: 26 under highly stringent conditions; The method of claim 2.   3. The method of claim 2, wherein the cells are stem cells, intervertebral disc cells, nucleus pulposus cells, or annulus fibrosus cells.   2. The method of claim 1, wherein the cells are transfected ex vivo.   2. The method of claim 1, wherein the cells are transfected in vivo.   3. The method of claim 2, wherein the cells are transfected in vivo by directly injecting the nucleic acid into a mammalian intervertebral disc.   2. The method of claim 1, wherein the nucleic acid is in a vector.   9. The method of claim 8, wherein the vector is an expression vector.   10. The method of claim 9, wherein the expression vector is a plasmid.   9. The method of claim 8, wherein the vector is a virus.   12. The method of claim 11, wherein the virus is an adenovirus.   12. The method of claim 11, wherein the virus is a retrovirus.   13. The method of claim 12, wherein the adenovirus is AdHLMP-1.   2. The method of claim 1, wherein the promoter is a cytomegalovirus promoter.   2. The method of claim 1, wherein the proteoglycan is a sulfated glycosaminoglycan.   The method of claim 1, wherein the LIM mineralized protein is RLMP, HLMP-1, HLMP-1s, HLMP-2, or HLMP-3.   The method of claim 1, wherein the LIM mineralized protein is HLMP-1.   2. The method of claim 1, wherein the expression of LIM mineralized protein increases the expression of one or more bone morphogenetic proteins in the cell.   A non-osseous mammalian cell comprising an isolated nucleic acid sequence encoding a LIM mineralized protein.   21. The non-osseous mammalian cell of claim 20, which is a stem cell, annulus fibrosus cell, nucleus pulposus cell, or intervertebral disc cell.   21. The non-osseous mammalian cell of claim 20, which is a pluripotent stem cell or mesenchymal stem cell. In a mammal, a method of treating intervertebral disc injury or disease comprising:
Transfecting the isolated nucleic acid into a mammalian cell capable of producing proteoglycan and / or collagen;
The isolated nucleic acid comprises a nucleotide sequence encoding a LIM mineralized protein operably linked to a promoter, and expression of the LIM mineralized protein stimulates proteoglycan and / or collagen synthesis in the cell , Said method.   24. The method of claim 23, wherein the disc degeneration is reversed, prevented or prevented.   24. The method of claim 23, wherein the disc disease is degenerative disc disease, lower back pain, disc herniation, or spinal stenosis.   24. The method of claim 23, wherein the cells are intervertebral disc cells and the cells are transfected in vivo by direct injection of the isolated nucleic acid into a mammalian intervertebral disc.   24. The method of claim 23, wherein the mammalian cells are transfected ex vivo and then transplanted into the mammal.   24. The method of claim 23, wherein the mammalian cell is a non-osseous mammalian cell.   28. The method of claim 27, wherein the mammalian cells are stem cells, intervertebral disc cells, annulus fibrosus cells, or nucleus pulposus cells.   28. The method of claim 27, wherein the mammalian cell is a pluripotent stem cell or a mesenchymal stem cell.   24. The method of claim 23, wherein the isolated nucleic acid is in a vector.   24. The method of claim 23, wherein the isolated nucleic acid is in an expression vector.   24. The method of claim 23, wherein the isolated nucleic acid is in a plasmid.   24. The method of claim 23, wherein the isolated nucleic acid is in a virus.   24. The method of claim 23, wherein the isolated nucleic acid is in an adenovirus.   24. The method of claim 23, wherein the isolated nucleic acid is in a retrovirus.   36. The method of claim 35, wherein the adenovirus is AdHLMP-1.   24. The method of claim 23, wherein the promoter is a cytomegalovirus promoter.   24. The method of claim 23, wherein the LIM mineralized protein is RLMP, HLMP-1, HLMP-1s, HLMP-2, or HLMP-3.   24. The method of claim 23, wherein the LIM mineralized protein is HLMP-1. Isolated nucleic acid:
Hybridizes to nucleic acid molecules complementary to the full length of SEQ ID NO: 25 under standard conditions; and / or hybridizes to nucleic acid molecules complementary to the full length of SEQ ID NO: 26 under highly stringent conditions; 24. The method of claim 23.   28. The method of claim 27, wherein the cells are transplanted into an intervertebral disc.   28. The method of claim 27, further comprising combining the transfected cells with a carrier prior to transplantation into the mammal.   44. The method of claim 43, wherein the carrier comprises a porous matrix.   45. The method of claim 44, wherein the carrier comprises a synthetic polymer or collagen matrix. An intervertebral disc implant comprising a carrier material; and a plurality of mammalian cells comprising an isolated nucleic acid sequence encoding a LIM mineralized protein;
The intervertebral disc implant, wherein the carrier material comprises a porous matrix of biocompatible material, and the mammalian cells are incorporated into the carrier.   49. The implant of claim 46, wherein the mammalian cells are selected from the group consisting of stem cells, annulus fibrosus cells, nucleus pulposus cells, intervertebral disc cells, and combinations thereof.   48. The implant of claim 46, wherein the mammalian cell is a pluripotent stem cell, a mesenchymal stem cell, or a combination thereof.   49. The implant of claim 46, wherein the biocompatible material comprises a synthetic polymer or protein.   48. The implant of claim 46, wherein the biocompatible material comprises collagen.

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