# Patent

## Title
NELL-1 peptide

## Abstract

The invention generally relates to a bone growth factor, and more particularly to compositions including NELL1, articles of manufacture including NELL1 and methods of using NELL1 to induce bone formation. This invention also provides methods for the expression and purification of NELL1 and NELL2 peptides.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This is a continuation application of U.S. application Ser. No. 10/544,553, filed on May 15, 2006, now issued as U.S. Pat. No. 7,544,486, which is a U.S. national phase of PCT/US2004/003808, filed on Feb. 9, 2004, which claims the benefit of U.S. provisional application No. 60/445,672, filed on Feb. 7, 2003. The teachings in these applications are incorporated herein in their entirety by reference.


STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This invention was made with Government support under Grant No. DE000422 and DE014649 awarded by the National Institutes of Health. The Government has certain rights in this invention.


FIELD OF THE INVENTION
The invention generally relates to a bone growth factor, and more particularly to compositions including NELL1, articles of manufacture including NELL1 and methods of using NELL1 to induce bone formation. This invention also provides methods for the expression and purification of NELL1 and NELL2 peptides.
BACKGROUND OF THE INVENTION
Growth factors are substances, such as peptides, which affect the growth and differentiation of defined populations of cells in vivo or in vitro.
Bone formation occurs during development of long bones (endochondral bone formation) and flat bones (intramembraneous bone formation). Further, bone formation occurs during bone remodeling which occurs continuously in adult life in order to preserve the integrity of the skeleton. Finally, bone formation occurs during bone repair, such as when bone wounds occur in a fracture or surgical situation, for example. While separate bone formation mechanisms are thought to be involved in the embryological development of long and flat bones and repair is thought to involve intramembraneous bone formation.
Bone formation by either mechanism involves the activity of osteoblasts, which are regulated by growth factors. Osteoblasts are derived from a pool of marrow stromal cells (also known as mesenchymal stem cells; MSC). These cells are present in a variety of tissues and are prevalent in bone marrow stroma. MSC are pluripotent and can differentiate into a variety of cell types including osteoblasts, chondrocytes, fibroblasts, myocytes, and adipocytes. Growth factors are thought to impact osteogenic cell proliferation, differentiation and osteoblast mineralization, each of which impacts bone formation.
Autogenous bone has been used, such to repair bone in patients with craniosynostosis and cleft grafting, for example. Craniosynostosis (CS), the premature closure of cranial sutures, affects 1 in 3,000 infants and therefore is one of the most common human congenital craniofacial deformities. Premature suture closure results in cranial dimorphism, which may need surgical correction. Premature suture closure in human CS may occur by two possibly distinct processes: calvarial overgrowth and bony fusion. Recently, FGF2 and FGFR1 have been implicated in premature cranial suture fusion via CBFA1-mediated pathways (8). Missense mutation of CBFA1 is linked to cleidocranial dysplasia, manifested as delayed suture closure.
Autologous bone grafting procedures have been performed utilizing autogenous bone, such as from the iliac crest or calvaria. These donor sites are not without associated morbidity including pain, gait disturbance, thigh paresthesia for iliac crest donor sites, and infection, neurologic deficits, and hematomas for calvarial grafts. Further, donor sites may have limited volume and may contribute to increased surgical time and hospital stay.
Alloplastic grafting materials have also been utilized, and growth factors have been tested in animal models. For example, bFGF has shown potential for use in bone regeneration and repair. Another family of osteogenic growth factors have been described as bone morphogenic protein (BMP). Specifically, BMP-2 recombinant protein has been demonstrated to regenerate mandibular continuity defects and cleft palate defects with results equal to or better than autogenous particulate bone and marrow. BMPs and other osteogenic factors have been studied for use in clinical applications. However, the cost of using minimally effective dosages of BMP has been a limiting factor in clinical use.
Spinal fusion is a surgical technique in which one more of the vertebrae of the spine are united together so that motion no longer occurs between them. Indications include: treatment of a fractured (broken) vertebra, correction of deformity, elimination of pain from motion, treatment of instability, and treatment of some cervical disc herniations. The surgery may involve placement of a bone graft between the vertebrae to obtain a solid union between the vertebrae. The procedure also may involve supplemental treatments including the placement of plates, screws, cages, and recently bone morphogenic protein 2 and 7 to assist in stabilizing and healing the bone graft. Autogenous bone grafting has been the clinically preferred method, and yet has about a 30-50% failure rate. Autogenous bone grafting is a separate surgery and also carries significant morbidity.
Therefore, safe, effective and affordable compositions and methods are desired to induce bone formation in bone development, disorders, or bone trauma.
SUMMARY OF THE INVENTION
This invention may provide methods for the expression and purification of NELL1 and NELL2 peptides. In one embodiment, the method includes NELL peptides, nucleic acid constructs expressing NELL peptides, and cells expressing NELL peptides which may be useful in producing quantities of NELL peptides. In one embodiment, the nucleic acid constructs expressing NELL peptides may further include nucleic acid sequences encoding signal peptides which may facilitate the protein trafficking and post production modification of the NELL peptides in the host cell. In one embodiment, the signal peptide may facilitate the secretion of the peptide from the host cell. Therefore, this invention is advantageous at least in providing quantities of functional NELL peptides which may be purified for clinical or research use.
The invention may include compositions and substrates including NELL peptides. In some embodiments, a composition may include NELL1, and may include additional agents which may effect the application, stability, activity, diffusion and/or concentration of the peptide relative to the application site, for example. In some embodiments, a substrate may include cells and/or NELL1 peptide which may facilitate bone repair in the proximity of the implant.
The invention may include methods of inducing osteogenic differentiation, osteoblastic mineralization and/or bone formation in a variety of clinical applications.
This invention is advantageous at least in that NELL peptides may provide a greater effect than known growth factors or may enhance the activity of other growth factors. Therefore, lower doses of each growth factor may be used for clinical applications. This is significant at least in that clinical treatments may be more affordable. Further this invention is advantageous at least in that NELL1 enhances osteogenic differentiation, osteoblastic mineralization and bone formation, which may improve the clinical rate and effectiveness of treatment with BMP alone.
DEFINITIONS
The terms “polypeptide”, “peptide” and “protein” may be used interchangeably herein to refer to a polymer of amino acid residues. The terms may apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
The terms “NELL1 cDNA” may refer to SEQ ID NO:1, 3 and 5 (FIGS. 1, 3 & 5 respectively), and “NELL2 cDNA” may refer to SEQ ID NO:7, 9, 11 and 13 (FIGS. 7, 9, 11 & 13).
A NELL1 peptide is a protein which may be expressed by the NELL1 gene or cDNA and includes SEQ ID NO: 2, 4, and 6 (FIGS. 2, 4 & 16, respectively). The NELL1 peptide may include a NELL1 peptide fragment that retains the ability to induce osteogenic cell differentiation, osteoblast differentiation or bone formation. A NELL2 peptide is a protein which may be expressed by the NELL2 gene or cDNA and includes SEQ ID NO: 8, 10, 12 and 14 (FIGS. 8, 10, 12 and 14, respectively). The NELL2 peptide may include NELL2 peptide fragments that retain similar activity to the full NELL2 peptide sequence.
The term “antibody” may include various forms of modified or altered antibodies, such as an intact immunoglobulin, an Fv fragment containing only the light and heavy chain variable regions, an Fv fragment linked by a disulfide bond, a Fab or (Fab)′2 fragment containing the variable regions and parts of the constant regions, a single-chain antibody and the like. An antibody may include intact molecules as well as fragments thereof, such as, Fab and F(ab′)2′, and/or single-chain antibodies (e.g. scFv) which may bind an epitopic determinant. An antibody may be of animal (such as mouse or rat) or human origin or may be chimeric or humanized. Antibodies may be polyclonal or monoclonal antibodies (“mAb's”), such as monoclonal antibodies with specificity for a polypeptide encoded by a NELL1 or NELL 2 protein.
The term “capture agent” may refer to molecules that specifically bind other molecules to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, and the like.
The term “specifically binds” may refer to a biomolecule (e.g., protein, nucleic acid, antibody, etc.), refers to a binding reaction which is determinative of the presence biomolecule in heterogeneous population of molecules (e.g., proteins and other biologics). Thus, under designated conditions (e.g. immunoassay conditions in the case of an antibody or stringent hybridization conditions in the case of a nucleic acid), the specified ligand or antibody may bind to its particular “target” molecule and may not bind in a significant amount to other molecules present in the sample.
The terms “nucleic acid” or “oligonucleotide” may refer to at least two nucleotides covalently linked together. A nucleic acid of the present invention may be single-stranded or double stranded and may contain phosphodiester bonds, although in some cases, nucleic acid analogs may be included that may have alternate backbones, comprising, for example, phosphoramide, phosphorothioate, phosphorodithioate, omethylphosphoroamidite linkages, and/or peptide nucleic acid backbones and linkages. Analog nucleic acids may have positive backbones and/or non-ribose backbones. Nucleic acids may also include one or more carbocyclic sugars. Modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments, for example.
The term “specific hybridization” may refer to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions, including conditions under which a probe may hybridize preferentially to its target subsequence, and may hybridize to a lesser extent to other sequences.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1D show a nucleic acid sequence encoding human NELL 1 cDNA (SEQ ID NO:1) and an amino acid sequence encoding human NELL 1 (SEQ ID NO:2).
FIGS. 2A-2B show an amino acid sequence encoding human NELL 1 (SEQ ID NO:2).
FIGS. 3A-3D show a nucleic acid sequence encoding rat NELL 1 cDNA (SEQ ID NO:3) and an amino acid sequence encoding rat NELL 1 (SEQ ID NO-4).
FIGS. 4A-4B show an amino acid sequence encoding rat NELL 1 (SEQ ID NO:4).
FIGS. 5A-5D show a nucleic acid sequence encoding mouse NELL 1 cDNA (SEQ ID NO:5) and an amino acid sequence encoding mouse NELL 1 (SEQ ID NO:6).
FIGS. 6A-6B show an amino acid sequence encoding mouse NELL 1 cDNA (SEQ ID NO:6).
FIGS. 7A-7D show a nucleic acid sequence encoding human NELL 2 cDNA (SEQ ID NO:7) and an amino acid sequence encoding human NELL 2 (SEQ ID NO:8).
FIGS. 8A-8B show an amino acid sequence encoding human NELL 2 (SEQ ID NO:8).
FIGS. 9A-9D show a nucleic acid sequence encoding rat NELL 2 cDNA (SEQ ID NO:9) and an amino acid sequence encoding rat NELL 2 (SEQ ID NO:10).
FIGS. 10A-10B show an amino acid sequence encoding rat NELL 2 (SEQ ID NO:10).
FIGS. 11A-11D show a nucleic acid sequence encoding mouse NELL 2 cDNA (SEQ ID NO:11) and an amino acid sequence encoding mouse NELL 2 (SEQ ID NO:12).
FIGS. 12A-12B show an amino acid sequence encoding mouse NELL 2 (SEQ ID NO:12).
FIGS. 13A-13D show a nucleic acid sequence encoding chicken NELL 2 cDNA (SEQ ID NO:13) and an amino acid sequence encoding chicken NELL 2 (SEQ ID NO:14).
FIGS. 14A-14B show an amino acid sequence encoding chicken NELL 2 (SEQ ID NO:14).
FIGS. 15A-15B show a flow diagram of one method of producing functional NELL peptide.
FIGS. 16A-16D illustrate a signal peptide-NELL1-FLAG nucleic acid construct (SEQ ID NO:15) and corresponding protein (SEQ ID NO:16). Underlined amino acid sequences are derived from melittin signal peptide. The bond between Alanine and Proline is a putative cleavage site for secretion by High Five cells. The residues from RTVLGFG---- (SEQ ID NO: 17, which is residues 38-44 of SEQ ID NO: 16) are derived from the mature protein of rat/human NELL 1 protein.
FIG. 17 illustrates the products of extracellular expression of NELL1-FLAG FIG. 17A is a CBB-stained SDS-PAGE gel of UnoQ-eluate containing purified NELL1 peptide produced from high five cells in serum-free medium (Productivity: ca. 3 mg/L medium); FIG. 17B is a Western blotting using anti-FLAG antibody. FIG. 17C is a CBB-stained SDS-PAGE gel of UnoQ-eluate containing purified NELL1 peptide produced from COS7 cells in serum-free medium (Productivity: <0.1 mg/L medium); FIG. 17D is a Western blotting using anti-FLAG antibody.
FIG. 18 is a Western blot illustrating the extracellular expression of NELL2-FLAG peptide by insect cells in serum-free medium.
FIG. 19 is a Western blot illustrating the extracellular expression of NELL1 and NELL2-FLAG peptides by high five cells in two types of serum free medium (Express Five SFM and ESF921).
FIG. 20 is a bar graph depicting alkaline phosphatase induction in fetal rat calvarial cells exposed to NELL1 peptide (1 ng, 10 ng, 100 ng/ml) and BMP4 (100 ng/ml).
FIG. 21A-D are photomicrographs of osteoblasts treated with NELL1 (A & B 5 ng/ml and C & D 50 ng/ml).
FIGS. 22A&B are photomicrographs of NELL1 MC3T3 micronodules forming micronodules in the absence of ascorbic acid; FIG. 22B is stained for alkaline phosphatase.
FIGS. 23A-C are photomicrographs depicting mineralization in A) anti-NELL, B) β-Gal and C) NELL adenoviral constructs; FIGS. 23D & E are bar graphs representing osteocalcin and osteoponin levels in each cell group over time.
FIG. 24 is a photomicrograph of a NELL1 over expressing transgenic mouse stained to depict mineralization demonstrating calvarial overgrowth.
FIGS. 25A & B are photomicrographs of calvaria stained for mineralization in A) NELL1 over expressing transgenic mouse and B) normal littermate, respectively.
FIG. 26 is a reverse transcriptase polymerase chain reaction blot depicting NELL1 gene expression in fetal rat calvarial cells treated with A) Cbfa1 or B) control.
FIG. 27A-C are photographs of skeletal staining of the cranium (top), clavicle (middle) and micro-CT of the cranium of A) wild-type, B) Cbfa1+/−, and C) Cbfa1+/−+NELL1overexp mice, respectively.
FIGS. 28A&B are photographs of microCT treated (right) and control (left) calvarial defects; A) BMP2 treated and B) NELL1 treated.
FIG. 29 is a photograph of microCT treated NELL1 (right) and BMP (left) calvarial defects.
FIGS. 30A&B are photographs of microCT treated NELL1 (right) and control (left) palatal defects.
FIGS. 31A&B are photomicrographs of TUNEL stained cartilage in A) NELL1overexp and B) wild type mice.
FIG. 32 is a flow diagram of one method of treating a patient to form bone in a selected location.
FIG. 33A is a schematic depicting one embodiment of an implant; FIG. 33B is a schematic depicting one embodiment of treating a patient to form bone in a selected location.



DETAILED DESCRIPTION
The present invention is related to agents and methods for inducing bone formation using NELL1. The present invention also is related to methods for the expression and purification of NELL1 and NELL2 proteins.
NELL1 was identified by Ting and Watanabe simultaneously. NELL1 is a 810 aa peptide, distributed primarily in bone. In adults, NELL2 is expressed at high levels in craniofacial bone, and lower levels in long bone. Its role in osteoblast differentiation, bone formation and regeneration has been examined. NELL2 was identified by Watanabe in 1996, and it is a 816 peptide, distributed in neural cells and brain.
Human NELL1 gene includes at least 3 Cbfa1 response elements in the promoter region. Cbfa1 specifically binds to these response elements in the NELL1 promoter. NELL1 expression may be under the control of this transcription factors expressed endogenously at least in preosteoblasts, osteoblasts and hypertrophic chondrocytes in development and in adulthood. Cleidocranial disostosis is a developmental cranial defect thought to be caused at least in part by Cbfa disruption.
In order to study the function of NELL1 and NELL2 peptides, attempts were made to produce and purify the peptide. Unfortunately, NELL1 and NELL2 peptides were unable to be expressed in a number of expression systems. Specifically, in E. coli direct and S. cerevisiae expression systems no expression was detected, in E. coli fused and CHO-dhfr expression systems, very low levels of expression occurred. In the baculovirus system, peptides were expressed.
It was a surprising discovery of this invention that NELL1 and NELL 2 peptides could be expressed at high levels in insect cells, and that the NELL1 and NELL2 peptides expressed in an insect system were functional forms of the protein.
COS7 cells can be used to produce NELL1 and NELL2 proteins at low levels, such as about 10 micrograms per litter medium, but require serum-containing medium for the expression. Unfortunately, this medium is not suitable for protein production. As for the signal peptides, NELL1 and NELL2 endogenous signal peptides permit peptide low levels of expression in COS7 cells.
In one embodiment, the invention includes a method of expressing a functional NELL peptide, such as NELL1 or NELL2 peptide, using an insect cell line. In one embodiment, the insect cell may be a high five cell, Sf9 and other Sf cells.
In one embodiment, the method may include providing a nucleic acid sequence encoding a NELL peptide, such as NELL1 or NELL2 peptide. The nucleic acid sequence may be a cDNA or genomic DNA, encoding at least a functional portion of a NELL peptide. For example, the nucleic acid sequence may be selected from the group including, but not limited to human NELL1 (SEQ ID NO:1), rat NELL1 (SEQ ID NO:3), mouse NELL1 (SEQ ID NO:5), or human NELL2 (SEQ ID NO:7), rat NELL2 (SEQ ID NO:9), mouse NELL2 (SEQ ID NO:11), chicken NELL2 (SEQ ID NO:13). The nucleic acid sequence may also include sequences such as those with substantial sequence similarity, such as sequences having at least about 75% sequence similarity with any portion of the sequences listed above.
Further the nucleic acid may include an expression vector for expressing the nucleic acid sequence encoding a NELL peptide, such as NELL1 or NELL2 peptide. For example, the expression vector may be pIZT/V5-His (Invitrogen), and selective markers may also include blastcidin and neomycin.
Further, the nucleic acid sequence may also include additional nucleic acids which encode reporter products to monitor levels of gene expression, or encode peptide tags which can be visualized using known methods in the art to monitor levels of peptide expression. Additional sequences may be selected so as to not interfere with the expression of the nucleic acid, or the functionality of the expressed peptide product.
In one embodiment, the method may include providing a nucleic acid sequence encoding a NELL peptide, such as NELL1 or NELL2 peptide, in frame with a nucleic acid sequence encoding a secretory signal peptide. In one embodiment, the secretory signal peptide may be a secretory signal peptide from a secreted bee protein. For example, the nucleic acid sequence may be selected from the group including, but not limited to a melittin signal sequence, drosphila immunoglobulin-binding protein signal sequence, equine interferon-gamma (eIFN-gamma) signal peptide, snake phospholipase A2 inhibitor signal peptide, human and/or chicken lysozyme signal peptide. For mammalian expression systems, a protrypsin leading sequence may also be used.
In one embodiment, the method may include transfecting an insect cell line with a nucleic acid construct encoding a NELL peptide; and culturing the insect cell line under conditions that permit expression and/or secretion of the NELL peptide. For example, the cell line may be transfected transiently or stably with the nucleic acid construct encoding a NELL peptide.
The method may also include collecting secreted NELL peptides and/or purifying NELL peptides for use. Peptide products may be tested for activity in a variety of functional or expression assays. For example in any assay, if a NELL peptide has a significant effect over a control substance on a given parameter, the NELL peptides may be said to be functional to effect the measured parameter.
In one embodiment, the invention may include a nucleic acid construct for expressing a NELL peptide, such as NELL1 and/or NELL2 peptide in an insect cell. The nucleic acid sequence may be a cDNA or genomic DNA, encoding at least a functional portion of a NELL peptide. For example, the nucleic acid sequence may be selected from the group including, but not limited to human NELL1 (SEQ ID NO:1), rat NELL1 (SEQ ID NO:3), mouse NELL1 (SEQ ID NO:5), or human NELL2 (SEQ ID NO:7), rat NELL2 (SEQ ID NO:9), mouse NELL2 (SEQ ID NO:11), chicken NELL2 (SEQ ID NO:13). The nucleic acid sequence may also include sequences such as those with substantial sequence similarity, such as sequences having at least about 75% sequence similarity with any portion of the sequences listed above.
The nucleic acid construct may include a nucleic acid sequence encoding a signal peptide. The nucleic acid may include an expression vector for expressing the nucleic acid sequence encoding a NELL peptide. Further, the nucleic acid sequence may include additional nucleic acids which encode reporter products to monitor levels of gene expression, or encode peptide tags which can be visualized using known methods in the art to monitor levels of peptide expression.
Nucleic acid constructs may comprise expression and cloning vectors should containing a selection gene, also termed a selectable marker, such as a gene that encodes a protein necessary for the survival or growth of a host cell transformed with the vector. The presence of this gene ensures that any host cell which deletes the vector will not obtain an advantage in growth or reproduction over transformed hosts. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate or tetracycline, (b) complement auxotrophic deficiencies.
Nucleic acid constructs may also include a promoter which is recognized by the host organism and is operably linked to the NELL encoding nucleic acid. Promoters are untranslated sequences located upstream from the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of nucleic acid under their control, including inducible and constitutive promoters. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g. the presence or absence of a nutrient or a change in temperature. At this time a large number of promoters recognized by a variety of potential host cells are well known.
A nucleic acid may be operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein which participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
In one embodiment, the invention may include cells that express functional NELL peptides. In one embodiment, the cell may be an insect cell. In one embodiment, the insect cell may be a high five cell.
In one embodiment, the cell may be transfected with a nucleic acid construct encoding a NELL peptide. For example, the cell line may be transfected transiently or stably with the nucleic acid construct encoding a NELL peptide. In one embodiment, NELL expressing nucleic acids (e.g., cDNA(s) may be cloned into gene expression vector or viral particles that are competent to transfect cells (such as insect cells).
The nucleic acid sequence may also include a nucleic acid sequence encoding a NELL peptide, such as NELL1 or NELL2 peptide, in frame with a nucleic acid sequence encoding an insect secretory signal peptide.
In one embodiment, the invention may include cells that express functional NELL peptides, and may secrete functional proteins.
In one embodiment, the invention may include a polypeptide (amino acid sequence) comprising a NELL peptide, such as NELL1 or NELL2 peptide, and may include secretory signal peptide.
For example, the amino acid sequence of the NELL peptide may be selected from the group including, but not limited to human NELL1 (SEQ ID NO:2), rat NELL1 (SEQ ID NO:4), mouse NELL1 (SEQ ID NO:6), or human NELL2 (SEQ ID NO:8), rat NELL2 (SEQ ID NO:10), mouse NELL2 (SEQ ID NO:12), chicken NELL2 (SEQ ID NO:14). The amino acid sequence may also include sequences such as those with substantial similarity, such as sequences having at least about 75% sequence similarity with any portion of the sequences listed above, or contain similar active binding domains as NELL1 peptides.
In one embodiment, the invention includes a method purifying NELL1 and/or NELL2 peptides secreted into culture media, according to standard peptide purification protocols, including, but not limited to those described below.
In one embodiment, whether a selected cell expresses a selected nucleic acid sequence to express and/or secrete a NELL peptide may be examined. In one embodiment, the presence, amount or and/or activity of NELL peptides may be examined.
In on embodiment, NELL peptides detected and quantified by any of a number of methods well known to those of skill in the art. These may include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, western blotting, and the like.
In one embodiment, Western blot (immunoblot) analysis may be used to detect and quantify the presence of NELL peptide(s) in a selected sample. This technique may include separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind a target peptide.
The assays of this invention may be scored (as positive or negative or quantity of target polypeptide) according to standard methods well known to those of skill in the art. The particular method of scoring may depend on the assay format and choice of label. For example, a Western Blot assay may be scored by visualizing the colored product produced by an enzymatic label. A clearly visible colored band or spot at the correct molecular weight may be scored as a positive result, while the absence of a clearly visible spot or band may be scored as a negative. The intensity of the band or spot may provide a quantitative measure of target polypeptide concentration.
The NELL1 proteins generated in such expression systems can be used in a manner analogous to the use of bone morphogenic proteins (e.g. BMP-1 through BMP-24). Thus, the NELL1 polypeptide(s) can be used to speed repair of bone fractures or to induce bone repair or replacement under circumstances where natural healing is limited or nonexistent. In addition, the NELL1 polypeptides can be incorporated into bone graft materials. These graft materials can be used in the treatment of fractures or to facilitate the replacement/healing of prostheses or bone transplants and spinal fusion.
The present invention may also include agents and methods for increasing the degree and/or rate of bone formation. More specifically, the invention may include the systemic and/or local application of agents for increasing bone formation. Clinical indices of a method or agents ability to increase the degree and/or rate of bone formation is evidenced by improvements in bone density at the desired site of bone formation as assessed by DEXA scanning. Enhanced bone formation in a healing fracture is routinely assessed by regular X-ray of the fracture site at selected time intervals. More advanced techniques for determining the above indices such as quantitative CT scanning may be used.
In one embodiment, the invention may include, a method of increasing osteogenic cell differentiation comprising increasing the concentration of a NELL1 gene product in an osteogenic cell, optionally applying a second agent; and inducing the expression of cellular marker of osteoblastic differentiation.
The method may include increasing the concentration of a NELL1 gene product by applying a NELL1 peptide to an osteogenic cell, and the NELL1 peptide may be selected from the group comprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6, or any portion of the NELL peptide which is effective in increasing osteoblastic differentiation. The method may include increasing the concentration of a NELL1 gene product by inducing the expression of an endogenous NELL1 gene, such as by increasing cellular levels of the expression regulating molecule, Cbfa1. The method may include increasing the concentration of a NELL1 gene product by transfecting the osteogenic cell with a nucleic acid construct encoding a NELL1 peptide, and the nucleic acid construct encoding a NELL1 peptide may be selected from the group comprising SEQ ID NO:1, SEQ ID NO: 3, or SEQ ID NO:5.
Osteogenic cells may include, but are not limited to osteoblasts, mesenchymal cells, fibroblasts, fetal embryonic cells, stem cells, bone marrow cells, dural cells, chondrocytes, chondroblasts and adipose stem cells.
Osteogenic cells may also include cells that are located within, are in contact with, or migrate towards (i.e., “home to”), bone tissue and which cells directly or indirectly stimulate the formation of bone tissue. As such, the osteogenic cells may be cells that ultimately differentiate into mature osteoblasts cells themselves, i.e., cells that “directly” form bone tissue.
A second agent may include, but is not limited to: TGF-β, BMP2, BMP4, BMP7, bFGF, collagen. The second agent may be selected to have a complimentary or synergistic effect with NELL1 in inducing osteoblastic differentiation.
Cellular markers of osteoblastic differentiation include, but are not limited to increased levels of alkaline phosphatase activity, osteocalcin and osteoponin mRNA expression, BMP7 expression, decorin expression and laminin B1 expression. However, any cellular marker whose activity or expression changes as a result of osteoblastic differentiation may be used as a marker of such.
In one embodiment, the method of increasing osteoblastic mineralization may include increasing the concentration of a NELL1 gene product in an osteoblastic cell, optionally applying a second agent; and inducing the expression of cellular marker of mineralization.
The method may include increasing the concentration of a NELL1 gene product by applying a NELL1 peptide to an osteogenic cell, and the NELL1 peptide may be selected from the group comprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6, or any portion of the NELL peptide which is effective in increasing osteoblastic mineralization. The second agent may be selected to have a complimentary or synergistic effect with NELL1 in inducing osteoblastic mineralization.
Cellular markers of osteoblastic mineralization include, but are not limited to increased levels of calcium incorporation. However, any cellular marker whose activity or expression changes as a result of osteoblastic mineralization may be used as a marker of such.
In one embodiment, a method of increasing intramembraneous bone formation may include increasing the concentration of a NELL1 gene product in a location where bone formation is desired, optionally applying a second agent to approximately the same location region where bone formation is desired; and inducing the formation of intramembraneous bone formation.
The method may include increasing the concentration of a NELL1 gene product by applying a NELL1 peptide to the location where bone formation is desired, and the NELL1 peptide may be selected from the group comprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6, or any portion of the NELL peptide which is effective in increasing intramembraneous bone formation.
The second agent may include, but is not limited to TGF-β, BMP2, BMP4, BMP7, bFGF, collagen, osteogenic cells, bone, bone matrix, tendon matrix, ligament matrix. The second agent may be selected to have a complimentary or synergistic effect with NELL1 in inducing intramembraneous bone formation.
The formation of intramembraneous bone may be evaluated by microscopic inspection for histology, DEXA scanning, X-ray or CT scanning of bone density in the area where bone formation is desired.
In one embodiment, a method of increasing endochondral bone formation may include increasing the concentration of a NELL1 gene product in a region where bone formation is desired; optionally applying a second agent to the region where bone formation is desired and at least inducing hypertrophy of chondroblast in the region where bone formation is desired.
The method may include increasing the concentration of a NELL1 gene product by applying a NELL1 peptide to the location where bone formation is desired, and the NELL1 peptide may be selected from the group comprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6, or any portion of the NELL peptide which is effective in increasing endochondral bone formation.
The second agent may include, but is not limited to TGF-β, BMP2, BMP4, BMP7, bFGF, collagen, osteogenic cells, bone, bone matrix, tendon matrix, ligament matrix. The second agent may be selected to have a complimentary or synergistic effect with NELL1 in inducing endochondral bone formation.
The formation of endochondral bone may be evaluated by chondroblast hypertrophy as viewed by an increase in hypertrophic and apoptotic chondroblasts, elucidated by TUNEL staining.
In one embodiment, the invention may include a method of incorporating NELL1 in carriers or substrates, and the resulting substrates.
In one embodiment, a composition for inducing bone formation may include an effective amount of a first agent to induce bone formation selected from the group including but not limited to a NELL1 peptide, and an agent that alters expression of NELL1 peptide, or an agent that alters the activity of a NELL1 peptide; and optionally a carrier.
The composition may include a NELL1 peptide selected from the group comprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6, or any fragment which is effective in inducing bone formation.
The composition may include a second agent including, but not limited to TGF-β, BMP2, BMP4, BMP7, bFGF, collagen, bone, bone matrix, tendon matrix or ligament matrix, osteogenic and/or osteoblastic cells.
In one embodiment, the carrier may be biodegradable, such as degradable by enzymatic or hydrolytic mechanisms. Examples of carriers include, but are not limited to synthetic absorbable polymers such as such as but not limited to poly(α-hydroxy acids) such as poly (L-lactide) (PLLA), poly (D, L-lactide) (PDLLA), polyglycolide (PGA), poly (lactide-co-glycolide (PLGA), poly (-caprolactone), poly (trimethylene carbonate), poly (p-dioxanone), poly (-caprolactone-co-glycolide), poly (glycolide-co-trimethylene carbonate) poly (D, L-lactide-co-trimethylene carbonate), polyarylates, polyhydroxybutyrate (PHB), polyanhydrides, poly (anhydride-co-imide), propylene-co-fumarates, polylactones, polyesters, polycarbonates, polyanionic polymers, polyanhydrides, polyester-amides, poly(amino-acids), homopolypeptides, poly(phosphazenes), poly (glaxanone), polysaccharides, and poly(orthoesters), polyglactin, polyglactic acid, polyaldonic acid, polyacrylic acids, polyalkanoates; copolymers and admixtures thereof, and any derivatives and modifications. See for example, U.S. Pat. No. 4,563,489, and PCT Int. Appl. # WO/03024316, herein incorporated by reference. Other examples of carriers include cellulosic polymers such as, but not limited to alkylcellulose, hydroxyalkylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, carboxymethylcellulose, and their cationic salts. Other examples of carriers include synthetic and natural bioceramics such as, but not limited to calcium carbonates, calcium phosphates, apatites, bioactive glass materials, and coral-derived apatites. See for example U.S. Patent Application 2002187104; PCT Int. Appl. WO/9731661; and PCT Int. Appl. WO/0071083, herein incorporated by reference.
In one embodiment, the carrier may further be coated by compositions, including bioglass and or apatites derived from sol-gel techniques, or from immersion techniques such as, but not limited to simulated body fluids with calcium and phosphate concentrations ranging from about 1.5 to 7-fold the natural serum concentration and adjusted by various means to solutions with pH range of about 2.8-7.8 at temperature from about 15-65 degrees C. See, for example, U.S. Pat. Nos. 6,426,114 and 6,013,591; and PCT Int. Appl. WO/9117965 herein incorporated by reference.
Other examples of carriers include, collagen (e.g. Collastat, Helistat collagen sponges), hyaluronan, fibrin, chitosan, alginate, and gelatin. See for example, PCT Int. Appls. WO/9505846; WO/02085422, herein incorporated by reference.
In one embodiment, the carrier may include heparin-binding agents; including but not limited to heparin-like polymers e.g. dextran sulfate, chondroitin sulfate, heparin sulfate, fucan, alginate, or their derivatives; and peptide fragments with amino acid modifications to increase heparin affinity. See for example, Journal of Biological Chemistry (2003), 278(44), p. 43229-43235, herein incorporated by reference.
In one embodiment, the substrate may be in the form of a liquid, solid or gel.
In one embodiment, the substrate may include a carrier that is in the form of a flowable gel. The gel may be selected so as to be injectable, such as via a syringe at the site where bone formation is desired. The gel may be a chemical gel which may be a chemical gel formed by primary bonds, and controlled by pH, ionic groups, and/or solvent concentration. The gel may also be a physical gel which may be formed by secondary bonds and controlled by temperature and viscosity. Examples of gels include, but are not limited to, pluronics, gelatin, hyaluronan, collagen, polylactide-polyethylene glycol solutions and conjugates, chitosan, citosan & b-glycerophosphate (BST-gel), alginates, agarose, hydroxypropyl cellulose, methyl cellulose, polyethylene oxide, polylactides/glycolides in N-methyl-2-pyrrolidone. See for example, Anatomical Record (2001), 263(4), 342-349, herein incorporated by reference.
In one embodiment, the carrier may be photopolymerizable, such as by electromagnetic radiation with wavelength of at least about 250 nm. Example of photopolymerizable polymers include polyethylene (PEG) acrylate derivatives, PEG methacrylate derivatives, propylene fumarate-co-ethylene glycol, polyvinyl alcohol derivatives, PEG-co-poly(-hydroxy acid) diacrylate macromers, and modified polysaccharides such as hyaluronic acid derivatives and dextran methacrylate. See for example, U.S. Pat. No. 5,410,016, herein incorporated by reference.
In one embodiment, the substrate may include a carrier that is temperature sensitive. Examples include carriers made from N-isopropylacrylamide (NiPAM), or modified NiPAM with lowered lower critical solution temperature (LCST) and enhanced peptide (e.g. NELL1) binding by incorporation of ethyl methacrylate and N-acryloxysuccinimide; or alkyl methacrylates such as butylmethacrylate, hexylmethacrylate and dodecylmethacrylate. PCT Int. Appl. WO/2001070288; U.S. Pat. No. 5,124,151 herein incorporated by reference.
In one embodiment, where the carrier may have a surface that is decorated and/or immobilized with cell adhesion molecules, adhesion peptides, and adhesion peptide analogs which may promote cell-matrix attachment via receptor mediated mechanisms, and/or molecular moieties which may promote adhesion via non-receptor mediated mechanisms binding such as, but not limited to polycationic polyamino-acid-peptides (e.g. poly-lysine), polyanionic polyamino-acid-peptides, Mefp-class adhesive molecules and other DOPA-rich peptides (e.g. poly-lysine-DOPA), polysaccharides, and proteoglycans. See for example, PCT Int. Appl. WO/2004005421; WO/2003008376; WO/9734016, herein incorporated by reference.
In one embodiment, the carrier may include comprised of sequestering agents such as, but not limited to, collagen, gelatin, hyaluronic acid, alginate, poly(ethylene glycol), alkylcellulose (including hydroxyalkylcellulose), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, blood, fibrin, polyoxyethylene oxide, calcium sulfate hemihydrate, apatites, carboxyvinyl polymer, and poly(vinyl alcohol). See for example, U.S. Pat. No. 6,620,406, herein incorporated by reference.
In one embodiment, the carrier may include surfactants to promote NELL1 stability and/or distribution within the carrier materials such as, but not limited to polyoxyester (e.g. polysorbate 80, polysorbate 20 or Pluronic F-68).
In one embodiment, the carrier may include buffering agents such as, but not limited to glycine, glutamic acid hydrochloride, sodium chloride, guanidine, heparin, glutamic acid hydrochloride, acetic acid, succinic acid, polysorbate, dextran sulfate, sucrose, and amino acids. See for example, U.S. Pat. No. 5,385,887, herein incorporated by reference. In one embodiment, the carrier may include a combination of materials such as those listed above.
By way of example, the carrier may a be PLGA/collagen carrier membrane. The membrane may be soaked in a solution including NELL1 peptide.
In one embodiment, an implant for use in the human body may include a substrate including NELL1 in an amount sufficient to induce bone formation proximate to the implant.
In one embodiment, an implant for use in the human body may include a substrate having a surface including NELL1 in an amount sufficient to induce bone formation proximate to the implant.
In one embodiment, an implant for use in the human body may include a substrate having a surface including osteogenic cells, and NELL1 in an amount sufficient to induce bone formation. In one embodiment, the implant may be seeded with cells, including but not limited to autologous cells, osteogenic or osteoblastic cells, cells expressing NELL1 or another osteogenic molecule.
An implant may include a substrate formed into the shape of a mesh, pin, screw, plate, or prosthetic joint. By way of example, a substrate may be in a form of a dental or orthopedic implant, and NELL1 may be used to enhance integration in bone in proximity to the implant. An implant may include a substrate that is resorbable, such as a substrate including collagen.
In one example, a composition according to this invention may be contained within a time release tablet.
The NELL1 peptide may be combined with a acceptable carrier to form a pharmacological composition. Acceptable carriers can contain a physiologically acceptable compound that acts, for example, to stabilize the composition or to increase or decrease the absorption of the agent. Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the anti-mitotic agents, or excipients or other stabilizers and/or buffers.
Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. One skilled in the art would appreciate that the choice of a carrier, including a physiologically acceptable compound depends, for example, on the route of administration.
The compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable may include powder, tablets, pills, capsules.
The compositions of this invention may comprise a solution of the NELL1 peptide dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier for water-soluble peptides. A variety of carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
The concentration of NELL1 peptide in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.
The dosage regimen will be determined by the clinical indication being addressed, as well as by various patient variables (e.g. weight, age, sex) and clinical presentation (e.g. extent of injury, site of injury, etc.).
However, a therapeutically effective dose of a NELL1 peptide or agent useful in this invention is one which has a positive clinical effect on a patient or desired effect in cells as measured by the ability of the agent to enhance osteoblastic differentiation, mineralization, bone formation, as described above. The therapeutically effective dose of each peptide or agent can be modulated to achieve the desired clinical effect, while minimizing negative side effects. The dosage of the peptide or agent may be selected for an individual patient depending upon the route of administration, severity of the disease, age and weight of the patient, other medications the patient is taking and other factors normally considered by an attending physician, when determining an individual regimen and dose level appropriate for a particular patient.
Dosage Form. The therapeutically effective dose of an agent included in the dosage form may be selected by considering the type of agent selected and the route of administration. The dosage form may include a agent in combination with other inert ingredients, including adjutants and pharmaceutically acceptable carriers for the facilitation of dosage to the patient, as is known to those skilled in the pharmaceutical arts.
In one embodiment, the invention may include a method of treating a patient to induce bone formation, comprising administering NELL1 peptide at a therapeutically effective dose in an effective dosage form at a selected interval to enhance bone formation. The method of may further comprise administering at least one secondary agent in the region where bone formation is desired, including but not limited to TGF-β, BMP2, BMP4, BMP7, bFGF, collagen, bone, bone matrix, tendon matrix or ligament matrix, osteogenic or osteoblastic cells.
In one embodiment, a method of treating a patient to induce bone formation may include harvesting mammalian osteogenic cells, increasing the concentration of expression of NELL1 peptide in contact with the osteogenic cells and administering the osteogenic cells to a region where bone formation is desired.
In one embodiment, bone formation to repair to cranial trauma or cranial defects may be desired, such as occurs in fetuses, infants or adults having cleidocranial disostosis, or cleft palate. In one embodiment, bone formation may be desired in a region of a non-healing bone defect (also known as critical size defect where bone fails to regenerate/heal in the defect). Critical size defect models are studied as a stringent test on agent effecting all bone healing, including long bone fracture, since all bone wound healing is believed to be by membranous (also called intramembraneous) bone formation. For example, long bone fracture and calvarial defect both heal by membranous bone formation. In one embodiment, bone formation may be desired in alveolar bone grafts or alveolar ridge augmentation, or periodontal bone defect. In one embodiment, bone formation may be desired to enhance the integration of implants such as joint or dental implants, or cosmetic surgery on plants.
In one embodiment, bone formation may be used in alternative or in addition to autologous, autogenous or alloplastic materials for bone grafts.
EXAMPLES
The following examples are offered to illustrate, but not to limit the claimed invention.
Example 1
Expression of NELL Peptides
A cDNA fragment was ligated into the expression vector PiZT/V5-His (3.4 kb) (EcoRV site, Invitrogen) and included a melittin signal peptide, BamHI-EcoRI cDNA fragment of the mature rat NELL1 and a FLAG tag sequence. FIG. 16 is a depiction of the nucleic acid sequence of the cDNA construct used in this example, and corresponding predicted peptide sequence.
The High five cells (BTI-TN-5B1-4) were adapted to serum-free medium, and cells were transfected with the NELL1 peptide expression vector. Cells were treated with zeocin so as to select only cell populations expressing the NELL1 FLAG constructs. Surviving cell populations were confirmed to be stable transformants. Extracellular media was collected and tested for the presence of NELL1 peptide. NELL1 peptide was purified and used in functional assays described below.
FIG. 17A is an illustration of a CBB-stained SDS-PAGE gel of UnoQ-eluate containing purified NELL1 peptide. The medium was applied onto UnoQ column (Bio-Rad) as described herein. FIG. 4B is an illustration of a Western blot using anti-FLAG antibody depicting NELL1-FLAG expression in reference to a protein ladder. Peptide: 140 kDa (intracellular precursor), 130 kDa (mature form; 90 kDa peptide), 400 kDa (secreted form, homotrimer). In the example above, the productivity of the expression system was about 3 mg NELL1 peptide/L medium.
Relative to other expression systems which did not express or secrete peptide at all (such as bacterial expression, including yeast) or whose peptide production was extremely low (e.g., E. coli fused peptide system, CHO-dhfr cells, >10 mcg/L) production with the systems described (mammalian and insect cells) was surprisingly and substantially more effective at producing large amounts of functional protein.
Expression and Purification of Recombinant Rat NELL1 Protein. For production of the C-terminally FLAG-tagged NELL1 peptide by insect cells. A pIZT-NELL1-FLC plasmid was constructed by inserting the rat NELL1 cDNA fused to a FLAG epitope sequence derived from the pTB701-NELL1-FLC plasmid (Kuroda, BBRC) into insect expression vector pIZT/V5-His (Invitrogen). Furthermore, NELL1 original secretory signal sequence was replaced to honeybee mellitin signal sequence using PCR methods. High Five cells were purchased from Invitrogen, and were cultured in High Five Serum-Free Medium (Invitrogen). High Five cells were transfected with the pIZT-NELL1-FLC plasmid using FuGene6 (Roche). Forty-eight hours after transfection, cells were selected with 400 mg/ml of Zeocin (Invitrogen). Replace selective medium every 3 to 4 days until the stable expression cell line was established. NELL1 secretion was confirmed using immunoprecipitation and Western blot analyses. High five cells were found to express NELL1 peptides (140-kDa) in the culture medium.
The recombinant rat NELL1-FLC peptide was purified from the culture medium of Zeocin-resistant High Five cells by anion exchange chromatography using a UNO Q-1 column (Bio-Rad). NELL1 peptide was eluted at 500 mM NaCl.
For production of the C-terminally FLAG-tagged NELL1 peptide by COS7 cells, a pcDNA3.1-NELL1-FLC plasmid was constructed by inserting the rat NELL1 cDNA linked to a FLAG epitope sequence derived from the pTB701-NELL1-FLC plasmid into mammalian expression vector pcDNA3.1 (Invitrogen). COS7 cells were cultured in DMEM supplemented with 10% FBS. COS7 cells were transfected with the pcDNA3.1-NELL1-FLC using the endogenous NELL signal peptide plasmid and using electroporation method. Forty-eight hours after transfection, culture medium was subjected to immunoprecipitation and Western blot analyses for NELL1 peptide.
FIG. 17C is an illustration of a CBB-stained SDS-PAGE gel of UnoQ-eluate. including NELL1-FLAG. These expression studies showed that COS cells did not express functional NELL peptide, without modifying the N terminal of the NELL to increase secretion efficiency such as including a signal sequence. FIG. 17D is an illustration of a Western blot using anti-FLAG antibody depicting NELL1-FLAG expression.
Expression and Purification of Recombinant Rat NELL2 Protein. For production of the C-terminally FLAG-tagged NELL2 peptide by insect cells. A pIZT-NELL1-FLC plasmid was constructed by inserting the rat NELL2 cDNA fused to a FLAG epitope sequence derived from the pTB701-NELL2-FLC plasmid into insect expression vector pIZT/V5-His (Invitrogen). High Five cells were purchased from Invitrogen, and were cultured in High Five Serum-Free Medium (Invitrogen). High Five cells were transfected with the pIZT-NELL1-FLC plasmid using FuGene6 (Roche). Forty-eight hours after transfection, cells were selected with 400 mg/ml of Zeocin (Invitrogen). Selective media was replaced every 3 to 4 days, until the stable expression cell line was established. NELL2 expression was confirmed in culture medium was confirmed using immunoprecipitation and Western blot analyses. High five cells were found to express NELL2 peptides (140-kDa) in the culture medium.
The recombinant rat NELL2-FLC peptide was purified from the culture medium of Zeocin-resistant High Five cells by anion exchange chromatography using a UNO Q-1 column (Bio-Rad). NELL2-FLC peptide was eluted at 500 mM NaCl.
Example 2
Purification of NELL2 Protein from Culture Medium
High Five cells carrying pIZT-FLC-NELL2 were cultured for about three days in serum free culture medium (1 L). The culture medium was centrifuged at. 3000×g for 5 minutes and the supernatant was collected. PMSF was added to a final concentration of 1 mM. Saturated ammonium sulfate solution (80% saturation (v/v) was added and the solution kept at 4 degrees for 1 hour. The solution was centrifuged at 15000×g for 30 min. and precipitate collected. Precipitate was dissolved in 50 ml of 20 mM Tris-HCl (pH 8.0), 1 mm EDTA at 4 degree and applied onto an anion-exchange chromatography UnoQ column (6 ml, Bio-Rad) equilibrated in 20 mM Tris-HCl (pH 8.0), 1 mM EDTA at 4 degree (1 ml/min speed by FPLC (Amersham-Pharmacia). The column was thoroughly washed with the same buffer.
The binding protein was then eluted by the gradation from 0 M to 1.5 M NaCl in the same buffer. The NELL2-FLAG fractions were identified by Western blotting using anti-Flag M2 (Sigma) Ab. The positive fractions were collected into one tube. Final product was dialyzed in the seamless cellulose tube (Wako, cutoff MW 12000) against 1 L PBS for overnight at 4 degree. The product was stored at −70 degree.
The purity of the NELL2-FLAG peptide was examined by SDS-PAGE/CBB staining. FIG. 18 is an illustration of a CBB-stained SDS-PAGE gel of UnoQ-eluate containing purified NELL2 peptide. Column A depicts a peptide band at about 130 kDa was isolated from the cell medium. “IP” refers to the Anti-FLAG antibody used for the immunoprecipitation; “WB” refers to the Anti-FLAG antibody used for the Western blotting detection.
FIG. 19 is a blot illustrating the expression of NELL1 and NELL2 from Five SFM. “ESF921” refers to a commercial name of a serum-free medium; “Five SFM” refers to a commercial name of a medium. The constructs for the expression of both NELL proteins are similar to those described above.
Example 3
Increases in alkaline phosphatase activity is an early cellular marker of osteoblastic differentiation. In one study, fetal rat calvarial cells were grown in the presence of: NELL1 (1 ng/ml, 10 ng/ml, 10 ng/ml) produced using the methods described herein, or BMP4 (100 ng/ml) for duration of time. Alkaline phosphatase was assayed in each sample by conventional methods.
FIG. 20 is a bar graph depicting alkaline phosphatase induction as a function of treatment in rat calvarial cell cultures (“OD”=Optic density). Therefore, treatment with NELL1 was more potent than BMP4 in inducing osteoblast differentiation, as measured by alkaline phosphatase induction.
FIG. 21 are photomicrographs of rat calvarial cell cultures treated with NELL1. Treatment with NELL1 induced alkaline phosphatase activity and cell micronodule formation in the absence of ascorbic acid, which is an indication of osteoblastic differentiation and a precursor to bone formation.
Example 4
Alkaline phosphatase assay is an early cellular marker of osteoblastic differentiation. In one study, rat calvarial osteoblasts were grown on a 24 well plate. Wells were divided into groups including: NELL1, BMP2, NELL1/BMP2 and control (no peptide). Treatments included the application peptides at 10 ng/ml. Alkaline phosphatase was assayed in each sample by conventional methods.











TABLE 1





Time
NELL1
BMP2
NELL1/BMP
Control




















24
hr
134%
159%
210%
100%


3
days
154%
145%
189%
100%









Therefore, NELL1 and BMP have an additive effect on osteoblast differentiation, as measured by alkaline phosphatase activity relative to control or cells treated with single peptides alone.
Example 5
To investigate the effect of NELL1 expression on osteoblastic differentiation, bone related gene expression was evaluated in a microarray of MC3T3 cells at 3, 6 and 9 days post-infection with a NELL1 expressing construct relative to cells infected with β-gal expressing constructs.







TABLE 2







Expression levels over control cells.











Day 3
Day 6
Day 9



post-infection
post-infection
post-infection














Up regulated
NA
Osteocalcin 2.5
Decorin 2.2




BMP7 2.1
Osteocalcin 2.6





Laminin B1 2.0





BMP7 3.2





Osteopontin 3.5





Col 15alpha1 2.6









Several bone related genes in NELL1 transfected cells were expressed at levels at least two fold higher than the β-gal control transfected cells. Therefore, since cellular markers of late osteoblastic differentiation (such as osteocalcin and osteoponin) are up regulated, NELL1 expression and production enhanced osteoblastic differentiation.
Example 6
Micronodule formation, or the aggregation of a plurality of osteoblasts is an indication of osteoblastic differentiation and a precursor to bone formation. The process is thought to be regulated by ascorbic acid.
To investigate the effects of NELL1 on micronodule formation, MC3TC cells were transfected with a NELL1 encoding construct, and grown in the absence of ascorbic acid.
FIGS. 22 A&B are photomicrograph of MC3TC cells expressing NELL1 forming micronodules and stained for alkaline phosphatase (B). NELL1 expression induced alkaline phosphate induction, as well and micronodule formation. Therefore, NELL1 is active in cell micronodule formation, which is a precursor to bone formation, and NELL1 alone is sufficient to induce osteoblast differentiation.
Example 7
Mineralization, or the intracellular accumulation of calcium is an indication of osteoblastic differentiation and a precursor to bone formation. To investigate the effects of NELL1 mineralization, primary calvarial cells were transfected with an adenoviral NELL1 encoding construct or a control construct encoding β-gal, or an antisense NELL1 virus. Cells were subsequently examined by Von Kassa staining to detect the presence of intracellular calcium accumulation after 3, 6, 9 and 12 days in culture. This demonstrates NELL1 can accelerate bone mineralization.
FIGS. 23A-C are photomicrographs of calvarial cells treated with the A) antisense NELL1 virus, B) β-gal or C) NELL1. The control cells had a moderate amount of mineralization, NELL1 expressing cells had increased levels of mineralization, and in antisense NELL1 cells mineralization was inhibited. This “knock-out” study shows that NELL1 is required for osteoblast differentiation.
FIGS. 23 D&E are bar graphs depicting osteocalcin and osteoponin mRNA expression as a ratio relative to control GAPFH, after 3, 6, 9 and 12 days in culture. NELL1 expressing cells expressed significantly elevated levels of osteocalcin and osteoponin mRNA after 12 days. Therefore, NELL1 is active in inducing the expression of late cellular markers of osteoblastic differentiation and mineralization, which is a precursor to bone formation.
Example 8
Transgenic animal models have been used to examine the effect of NELL1 over expression on bone formation. CMV promoter was linked to NELL1 cDNA and microinjected into fertilized eggs. NELL1 was pan-over-expressed under potent CMV promoter.
FIG. 24 is a photomicrograph of a NELL1 transgenic mouse tissue, depicting Von Kassa staining. As shown, in FIG. 24 NELL1 transgenic mice had calvarial overgrowth, confirming NELL1's ability to induce bone growth including membranous bone formation.
FIGS. 25 A&B are photomicrographs depicting Von Kassa staining of calvaria of a NELL1 transgenic mouse (A) and normal littermate (B). As shown in FIG. 25A, NELL1 transgenic mice had enhanced mineralization relative to the normal littermate confirming NELL1's role in membranous bone formation.
Example 9
Transgenic animal models have been used to examine the effect of NELL1 expression on Cbfa1 deficiency induced developmental defects.
To determine whether Cbfa1 may play a role in NELL1 regulation, fetal rat calvarial cells were transfected with plasmid vectors containing mouse Cbfa1.
FIG. 26 is a blot depicting expression of NELL1 in Cbfa1 transfected cells at 24 and 48 hours relative to control cells. Cbfa1 transfection up regulated NELL1 expression within 24 hours (along either positive control osteocalcin). This shows NELL-1 is downstream of Cbfa1—a key “osteoblast transcription factor”.
FIG. 27A-C are photographs of skeletal staining (top, middle) and micro-CT (bottom). FIG. 27A depicts the normal skeletal pattern of a wild-type mouse. Typical boarders of mineralization are noted (dashed lines), anterior and posterior fontenelles (asterisks), and outline of the right coronal suture can be seen (arrows). Also, a normal clavicle is shown (A-middle). The micro-CT reveals the typical craniofacial bone morphology. FIG. 27B depicts skeletal defects of a Cbfa1+/− animal. Specifically, defective bone mineralization and bone formation is present in the poorly stained tissue (between the dotted lines) lateral to the midline calvarial defect, and lucency can also be seen in the area of the coronal structure (arrows). A significant degree of clavicle hypoplasia is noted (B-middle). Fig. DC depicts skeletal defects of a Cbfa1+/−+NELL1overexp animal demonstrating significantly increased calvarial bone formation relative to the Cbfa1+/− haploid deficient animal on skeletal staining and micro-CT. Also, a significantly lesser degree of clavicle hypoplasia relative to the Cbfa1+/− haploid deficient animal (middle). Note the restoration of bony overlap at the coronal sutures (arrows). Therefore, NELL1 over expression rescued Cbfa1 deficiency in transgenic mice confirming NELL1's role in membranous bone formation and endochondral bone formation. Further, NELL 1 can regenerate bone in bone in birth defects.
Example 10
Critical size defect is an important model for the study of an agents ability to induce intramembraneous bone repair. To investigate the effects of NELL1 on bone repair, right and left calvarial defects (3 mm) were created in wild-type adult CD-1 male mice. Left defects (control) were grafted with a PLGA/collagen carrier membrane only while right defects were grafted with PLGA/collagen carrier membrane soaked in either 200 ng of NELL1 or BMP2 per site. Calvaria were extracted and examined by microCT analysis.
FIG. 28A is a photograph of control (left) and BMP2 (right) treatment of calvarial defect; is a photograph of control (left) and NELL1 (right) treatment of calvarial defect; FIG. 29 is a photograph of NELL1 (left) and BMP2 (right) treatment of calvarial defect. Significant amount of bone formation was observed in both NELL1 and BMP2 groups. Therefore, NELL1 expression significantly effected bone formation and induce bone regeneration in the critical size defect model confirming NELL1's role in membranous bone formation.
Example 11
Rapid Palatal Expansion (RPE) is another model for the study of an agents ability to induce intramembraneous bone repair. To investigate the effects of NELL1 on bone repair, 4-week old Sprague Dawley rats were divided into groups for 1) control expansion, and 2) expansion with NELL1 treatment. The rats were sacrificed and their palates extracted an kept vital in organ culture. The palates were expanded and NELL1 added to the treatment group for 9 days.
FIGS. 30A&B are photographs of expanded palates treated with NELL1 (A) and control (B). Significant amount of bone formation was observed in both NELL1 and BMP2 groups. Therefore, NELL1 treatment significantly effected bone formation in the RPE model confirming NELL1's role in membranous bone formation.
Example 12
Endochondral bone formation is the key process in long bone development. It has several stages including: chondroblast proliferation, hypertrophy, apoptosis, invasion of blood vessel, replacement by osteoblasts. Acceleration of any one of these stages will induce endochrondral bone growth.
FIGS. 31A&B are photomicrographs of cartilage with TUNEL staining for apoptotic cells in NELL1 over expressing transgenic mice (A) and wild type mice (B).
As shown in FIG. 31A, in NELL1 over-expression in mice, cartilage shows hypertrophic chondroblasts and apoptosis (indicated by the brown staining using TUNEL ASSAY for identifying shrinkage of apoptotic nuclei). In FIG. 31B is a normal mouse (wild type) cartilage with TUNEL staining very few apoptotic cells are present and the cells are not hypertrophic. Therefore, NELL1 can induce cartilage hypertrophy and apoptosis, thereby inducing long bone formation and regeneration.
Example 13
NELL Substrate Preparation
In vitro. Polylactide-co-glycolide (85:15 PLGA; intrinsic viscosity  ̃0.6 dL/g, Birmingham Polymers, AL) was dissolved in chloroform to prepare 5% solution and poured into glass culture dishes and allowed to slowly evaporate for 24 hours. After solvent extraction, the films were coated according to the 8 groups below: (a) polymer only with no coating; (b) conventional apatite (1×SBF followed by 1.5×SBF); (c) accelerated biomimetic apatite (5×SBF followed by Mg-free and carbonate-free 5×SBF); (d) fibronectin (0.01 mg/ml); (e) poly-L-lysine (0.01 mg/ml); (f collagen; (g) Mefp-1 (0.01 mg/ml); and (h) mixture of collagen & hyaluronan. Each group was subdivided into NELL1 containing (100 ng) and NELL1-free groups, and cultured in vitro for 7 days with primary osteoblasts in non-differentiation media (no ascorbic acid, no beta glycerol phosphates). For each material, NELL1 groups stimulated higher alkaline phosphatase activity than NELL1 counterparts. Among the materials, accelerated apatites (group c) induced the greatest, and polymer control (group a) induced the least alkaline phosphatase activities.
In vivo. Polylactide-co-glycolide (85:15 PLGA; intrinsic viscosity  ̃0.6 dL/g, Birmingham Polymers, AL) was dissolved in chloroform and mixed with porogens (sucrose granules with diameter  ̃100-300 μm) to produce  ̃90% porosity PLGA scaffolds after particulate leaching and solvent extraction. Porous scaffolds were argon-plasma-etched, sterilized, coated with aqueous bovine type I collagen mixture containing 200 ng NELL1 peptide, dried, and implanted into calvarial defects of adult male wild-type mice. Positive control (PLGA/collagen/BMP) and negative controls (PLGA/collagen only; no growth factors), were also implanted into similar defects. At 4 week, microCT analysis show that while little or no bone formation was induced by the negative control scaffolds (PLGA/collagen only), NELL1-containing and BMP-containing scaffolds induced rapid and complete mineralization across the defects by week 4. Conventional histology confirmed that the mineralization presents the classic features of mature bone.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. It will be understood that the invention may also comprise any combination of the embodiments described or combination with known methods and compositions.
Although now having described certain embodiments of NELL peptide expression systems and bone formation activity of NELL peptide, it is to be understood that the concepts implicit in these embodiments may be used in other embodiments as well. In short, the protection of this application is limited solely to the claims that now follow.

## Claims

1. A polypeptide comprising a NELL1 peptide and a secretory signal peptide:
wherein the secretory signal peptide is selected from the group consisting of a melittin signal sequence, a drosphila immunoglobulin-binding protein signal sequence, an equine interferon-gamma (eIFN-gamma) signal peptide, a snake phospholipase A2 inhibitor signal peptide, a human lysozyme signal peptide, and a chicken lyzozyme signal peptide.

2. The polypeptide of claim 1, wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.