PERIOSTEAL SKELETAL STEM CELLS IN BONE REPAIR
20220096600 · 2022-03-31
Inventors
Cpc classification
C12N2501/21
CHEMISTRY; METALLURGY
C07K14/51
CHEMISTRY; METALLURGY
A61K38/1875
HUMAN NECESSITIES
A61P19/08
HUMAN NECESSITIES
A61K38/191
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
C07K14/523
CHEMISTRY; METALLURGY
C12N2533/40
CHEMISTRY; METALLURGY
C12N5/0654
CHEMISTRY; METALLURGY
C12N2501/155
CHEMISTRY; METALLURGY
A61K2300/00
HUMAN NECESSITIES
A61K38/1875
HUMAN NECESSITIES
A61L27/54
HUMAN NECESSITIES
A61L27/36
HUMAN NECESSITIES
C07K16/24
CHEMISTRY; METALLURGY
A61L2430/02
HUMAN NECESSITIES
A61K38/191
HUMAN NECESSITIES
A61K31/46
HUMAN NECESSITIES
International classification
A61L27/36
HUMAN NECESSITIES
A61P19/08
HUMAN NECESSITIES
C07K14/51
CHEMISTRY; METALLURGY
Abstract
Embodiments of the disclosure encompass methods and compositions for bone repair and bone injury healing. In some embodiments, the bone repair and bone injury healing utilizes the enhancement of migration of certain types of bone cells upon stimulation by a particular cytokine. In specific embodiments, migration of periosteal skeletal stem cells upon delivery of CCL5 and/or TNFα is enhanced and fosters bone repair and healing.
Claims
1. A method of inducing bone healing in an individual comprising administering at least one cytokine to the individual at a site in need thereof, wherein the cytokine is selected from the group consisting of CCL5, TNFα, BMP2, Wnt, and a combination thereof.
2. The method of claim 1, wherein administering of the cytokine comprises delivery directly to the site of the individual.
3. The method of claim 1, wherein administering comprises delivery of the cytokine with a gel and/or scaffold.
4. The method of claim 3, wherein the gel comprises matrigel, hydrogel, periosteum, hydroxyapatite, tricalcium phosphate, polystyrene, poly-1-lactic acid, polyglycolic acid, poly-dl-lactic-co-glycolic acid, collagen, proteoglycans, alginate-based substrates, chitosan, collagen-GAG, extracellular matrix, or a combination thereof.
5. The method of claim 1, wherein the administering comprises delivery of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 ng of cytokine to the site in need.
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7. The method of claim 1, wherein the delivery is by injection.
8. The method of claim 1, wherein the site in need comprises a bone fracture, bone break, bone degeneration, bone irradiation, a site of osteoporosis, or a combination thereof.
9. The method of claim 1, wherein the administering to the individual comprises administration of at least one cytokine and at least one additional therapy wherein the at least one cytokine and the at least one additional therapy are administered to the individual at the same time or at different times, wherein when they are administered at the same time, the at least one cytokine and the at least one additional therapy are administered to the individual in the same composition or in different compositions, optionally wherein the additional therapy comprises surgical repair, fixation of a device to set the site in need, placing the individual in a cast or sling, or a combination thereof.
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13. The method of claim 1, wherein the site in need is monitored for bone regeneration.
14. The method of claim 1, wherein the administration to the individual is prophylactic.
15. The method of claim 1, wherein the individual has osteoporosis, osteopetrosis, or osteosclerosis.
16. A method of recruiting periosteal skeletal stem cells to a site in an individual in need thereof, comprising administering at least one cytokine to the individual at the site in need, wherein the cytokine is selected from the group consisting of CCL5, TNFα, BMP2, Wnt, and a combination thereof.
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29. A method of reducing osteoclast migration to a site in need in an individual, comprising administering at least one cytokine to the individual at the site in need, wherein the cytokine is selected from the group consisting of CCL5, TNFα, BMP2, Wnt, and a combination thereof.
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43. A method of inhibiting bone formation at a site in need in an individual, comprising administering to an individual at least one agent that inhibits the function of CCR5 TNFα, BMP2, Wnt or a combination thereof.
44. The method of claim 43, wherein the agent is maraviroc or an antibody.
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50. The method of claim 43, wherein the site in need comprises a bone spur or ectopic bone formation site or bunion.
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57. A composition for administering to a bone site, comprising (a) a cytokine and/or agent, and (b) a gel and/or scaffold, wherein the cytokine and/or agent comprises a chemokine, an inflammatory cytokine, a bone morphogenetic protein, a Wnt protein, a CCR5 receptor antagonist, an antibody, or a combination thereof.
58. The composition of claim 57, wherein the cytokine and/or agent comprises CCL5, TNFα, BMP2, Wnt, maraviroc, an antibody that targets CCL5, an antibody that targets TNFα, an antibody that targets BMP2, an antibody that targets Wnt, an antibody that targets CCR5 or a combination thereof.
59. The composition of claim 57, wherein the gel and/or scaffold comprises matrigel, hydrogel, periosteum, hydroxyapatite, tricalcium phosphate, polystyrene, poly-1-lactic acid, polyglycolic acid, poly-dl-lactic-co-glycolic acid, collagen, proteoglycans, alginate-based substrates, chitosan, collagen-GAG, extracellular matrix, or a combination thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] For a more complete understanding of the present disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawings.
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DETAILED DESCRIPTION
I. Examples of Definitions
[0038] In keeping with long-standing patent law convention, the words “a” and “an” when used in the present specification in concert with the word comprising, including the claims, denote “one or more.” Some embodiments of the disclosure may consist of or consist essentially of one or more elements, method steps, and/or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.
[0039] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1%. With respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Unless otherwise stated, the term ‘about’ means within an acceptable error range for the particular value.
[0040] As used herein, the term “administer” or “administration” refers to providing a therapeutically effective amount of a composition to an individual at a site in need. The method of administration may or may not include injection, such as with a syringe, or delivery, such as implantation of the composition at the site in need. Other delivery routes are encompassed in the disclosure and detailed elsewhere herein.
[0041] When referring to “cells”, it is meant any number of cells, including a single cell, that is sufficient to induce or perform the disclosed method.
[0042] “Treatment,” “treat,” or “treating” means a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the disease or condition itself rather than just the symptoms. The treatment can be any reduction from pre-treatment levels and can be but is not limited to the complete ablation of the disease, condition, or the symptoms of the disease or condition. Therefore, in the disclosed methods, treatment” can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or the disease progression, including reduction in the severity of at least one symptom of the disease. For example, a disclosed method for reducing the immunogenicity of cells is considered to be a treatment if there is a detectable reduction in the immunogenicity of cells when compared to pre-treatment levels in the same subject or control subjects. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. It is understood and herein contemplated that “treatment” does not necessarily refer to a cure of the disease or condition, but an improvement in the outlook of a disease or condition. In specific embodiments, treatment refers to the lessening in severity or extent of at least one symptom and may alternatively or in addition refer to a delay in the onset of at least one symptom.
II. Methods of Enhancing Bone Formation
[0043] Particular embodiments of the disclosure are directed towards methods of administration of compositions to one or more sites in need, such as a bone, in an individual. The site in need may be a bone break, bone fracture, bone degeneration, site of bone irradiation, and so forth. The individual may have an injury, osteoporosis, osteopetrosis, osteosclerosis, or other bone degenerative diseases. In some embodiments, the site may be one that would benefit from prophylactic systems, methods, or compositions to delay the onset or inhibit bone degeneration partially or fully, for example. The individual may also be administered other conventional or novel methods and/or compositions to aid in the bone healing process. Such methods include, for example, surgery, placing the individual in a cast or sling, a fixation device, or a combination thereof.
[0044] In particular embodiments, the methods are directed towards modulation of specific skeletal stem cells (SSCs), including periosteal SSCs (P-SSCs), in an individual. The P-SSCs may be positive for the Mx1 marker. These P-SSC may have the ability to differentiate into osteoblasts, which can aid in repairing a site in need. The methods disclosed herein may induce migration of the P-SSCs to a specific site by administration of one or more compositions at the specific site(s). In particular embodiments, the recruitment of P-SSCs, including those that differentiate into osteoblasts, reduces the recruitment of cells that may be, or differentiate into, osteoclasts. In some embodiments, the methods are directed towards modulation of SSCs, including osteoclast progenitors (OCPs), which may express the CathespsinK marker. OCPs may have the ability to differentiate into osteoclasts, which can reduce the healing response at the site in need.
[0045] In particular embodiments, the method is directed to induce bone healing at a site in need comprising the administration of at least one composition, such as a cytokine and/or stem cell. The site in need may be a bone break, bone fracture, bone degeneration, site of bone irradiation, osteoporosis, or other bone degenerative diseases, or a site that would benefit from prophylactic systems, methods, or compositions to delay or stop bone degeneration, for example. Bone healing may be induced by recruitment of P-SSCs to the site in need, wherein the P-SSCs contribute to bone healing, such as by differentiating into osteoblasts, for example. The cytokine administered to the site in need may be CCL5, TNFα, BMP2, Wnt, or a combination thereof. On specific combination may be CCL5 and TNFα. The stem cell administered may be a P-SSC, including an Mx1.sup.+αSMA.sup.+ P-SSC, as one example. In some cases, the stem cell is administered with the one or more cytokines. In a specific aspect, a P-SSC, such as Mx1.sup.+αSMA.sup.+ P-SSC, is administered with CCL5, TNFα, BMP2, Wnt, or a combination thereof. The P-SSC may be administered with CCL5 and/or TNFα, in some cases.
[0046] In any method of the disclosure, the one or more cytokines and/or stem cells (including P-SSCs) are both provided to an individual for the purpose of enhancing bone formation and optionally may include an additional therapy for bone formation. In such cases, the one or more cytokines, the stem cells, and/or the additional therapy are administered to the individual at the same time or in succession. In cases wherein the one or more cytokines, the stem cells, and/or the additional therapy are administered to the individual in succession, the order of delivery may be in any suitable order. In some cases, the individual is administered the one or more cytokines before, during, or after the stem cells and/or additional therapy. In some cases, the individual is administered the stem cells before, during, or after the one or more cytokines and/or additional therapy. In some cases, the individual is administered the additional therapy before, during, or after the one or more cytokines and/or stem cells.
[0047] When the individual is administered CCL5, TNFα, BMP2, Wnt, or a combination thereof, the form of the CCL5, TNFα, BMP2, Wnt, or a combination thereof may be of any kind, including in polynucleotide form, such as on a vector of any kind (viral or non-viral), or in polypeptide form. In some cases, the CCL5, TNFα, BMP2, Wnt, or a combination thereof are provided in their full form, whereas in other cases the CCL5, TNFα, BMP2, Wnt, or a combination thereof are provided in a functional fragment or derivative; in such cases, a fragment or derive is function if after delivery it is able to enhance bone formation.
III. Methods of Inhibiting Bone Formation
[0048] In particular embodiments, an individual is in need of inhibiting bone formation, such as at a particular site. The inhibition of bone formation may be at the site of a bone spur (including heel spurs) or site of ectopic bone formation or bunion(s), for example. The bone spur may be caused by local inflammation, such as from degenerative arthritis (osteoarthritis) or tendinitis. The ectopic bone formation may be caused by surgery or trauma, such as to the hips and legs, for example from joint replacement (for example, from total hip arthroplasty) and/or a severe fracture of bone, such as the long bones of the lower leg. Such inhibition of bone formation may or may not include the induction of migration of cells that are osteoclasts or that differentiate into osteoclasts to a site in need of bone inhibition.
[0049] In other particular embodiments, the composition(s) to be administered comprises an agent that inhibits CCR5, such as maraviroc, for example. The inhibition of CCR5 may induce the migration of cells that are, or differentiate into, osteoclasts, such as OCPs, for example. The site in need in such methods may including sites of ectopic bone formation, such as in muscle or tendon tissue. Ectopic bone formation can be present at birth, be the result of genetics, or arise as a complication of certain medical conditions such as paraplegia and/or traumatic injury, as examples. The composition to be administered may comprise other materials, such as a biocompatible gel and/or scaffold, for example.
[0050] An individual in need of inhibition of bone formation may be determined to be in need of inhibition of bone formation, such as upon determination of ectopic bone formation or upon identification of a bone spur or bunion, or upon identification of a risk thereof.
IV. Cytokines and Inhibitors
[0051] In particular embodiments, the methods disclosed are directed to administration of a composition at a site in need, such as a bone, in an individual. The composition, in some embodiments, comprises at least one cytokine. The cytokine may be CCL5 and/or TNFα. The cytokine may be produced by any known means of obtaining cytokines, including recombinant protein purification, mammalian expression constructs, viral or non-viral transfections, isolation from cellular or hematopoietic sources, or purchasing from a commercial vendor, for example. The cytokine may also be produced in the individual from a polynucleotide or viral gene therapy. The composition may be delivered in a cell, in some cases (MSCs or isolated skeletal stem cells).
[0052] In particular embodiments, the cytokine comprises the CCL5 cytokine, wherein the CCL5 cytokine is translated from the CCL5 coding sequence, including as exemplary sequences, the sequences encoded by GenBank® Accession No NM_001278736.1 (SEQ ID NO:1) or NM_002985.2 (SEQ ID NO:2), or any homologous sequence from a human or non-human species that produces the same function as the exemplary sequence provided. In cases wherein the CCL5 cytokine is employed as a polypeptide, examples of the polypeptide include GenBank Accession No. NP_001265665.1 (SEQ ID NO:3) or NP_002976.2 (SEQ ID NO:4).
[0053] In particular embodiments, the cytokine comprises the TNFα cytokine, wherein the TNFα cytokine is translated from the TNFα coding sequence, including as an exemplary sequence, the sequence encoded by GenBank® Accession No NM_000594.3 (SEQ ID NO:5), or any homologous sequence from a human or non-human species that produces the same function as the exemplary sequence provided. In cases wherein the TNFα cytokine is employed as a polypeptide, an example of the polypeptide encoded by GenBank Accession No. NM_000594.3 (SEQ ID NO:6).
[0054] In particular embodiments, the cytokine comprises the BMP2 cytokine, wherein the BMP2 cytokine is translated from the BMP2 coding sequence, including as an exemplary sequence, the sequence encoded by GenBank® Accession No NM_001200.3 (SEQ ID NO:7), or any homologous sequence from a human or non-human species that produces the same function as the exemplary sequence provided. In cases wherein the BMP2 cytokine is employed as a polypeptide, an example of the polypeptide includes GenBank Accession No. NP_001191.1 (SEQ ID NO:8).
[0055] In particular embodiments, the cytokine comprises the Wnt cytokine, wherein the Wnt cytokine is translated from the Wnt coding sequence, including as an exemplary sequence, (Wnt3a in mouse bone injury is reported to induce bone healing and regeneration; Science Translational Medicine 28 Apr. 2010: Vol. 2, Issue 29, pp. 29ra30), or any homologous sequence from a human or non-human species that produces the same function as the exemplary sequence provided.
[0056] Homologous sequences may be sequences that have silent mutations, wherein the homologous sequence produces the same amino acid sequence. Homologous sequences may also, in a non-limiting example, be sequences that have mutations that code for amino acids that are functionally or practically equivalent to the amino acid coded in the exemplary sequence. Homologous sequences may also have insertions or deletions relative to the exemplary sequence, wherein the insertions or deletions do not alter the inherent function of the translated cytokine. Homologous sequences may also be from a non-human species that encode for a protein or polypeptide that is functionally equivalent to CCL5, TNFα, BMP2, or Wnt. Methods to produce cytokines that utilize non-natural amino acids, such as to enhance the function of the cytokine or improve the stability of the cytokine, may also be used. In certain embodiments, the cytokine may comprise a biologically active portion or fragment of any of the provided full length cytokines that may be administered to the individual. The fragment or portion may comprise approximately 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acids of the full length cytokine. Particular embodiments disclosed herein provide the use of at least one cytokine, protein, or polypeptide, or at least one polynucleotide encoding a cytokine, protein, or polypeptide, that functions equivalently to any one of the cytokines selected from the group consisting of CCL5, TNFα, BMP2, Wnt, or a combination thereof.
[0057] In cases where a CCL5, TNFα, BMP2, or Wnt polynucleotide is utilized in methods of the disclosure, the polynucleotide may be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:7, respectively. In embodiments where a CCL5, TNFα, BMP2, or Wnt polypeptide is utilized in methods of the disclosure, the polypeptide may be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3, SEQ ID NO:4, the polypeptide encoded by SEQ ID NO:6, or SEQ ID NO:8, respectively. In certain embodiments, the polypeptide may be a fragment of a polypeptide of SEQ ID NO:3, SEQ ID NO:4, the polypeptide encoded by SEQ ID NO:6, or SEQ ID NO:8, respectively; the fragment may be at least 25, 50, 75, 100, 125, 150, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, and so forth.
[0058] In particular embodiments, the composition administered to the site in need is an agent that inhibits CCR5, such as maraviroc, aplaviroc, vicriviroc, or related agents. The composition may also include an agent that inhibits the function of CCL5, TNFα, BMP2, or Wnt. The agents may be antibodies, including neutralizing antibodies, humanized antibodies, scFvs, or a combination thereof. Examples of antibodies that target CCR5 include those described by Olson and Jacobson (Curr Opin HIV AIDS, 2010) which is incorporated by reference herein in its entirety. Examples of antibodies that target CCL5 include those described by Aldinucci et al (International Journal of Cancer, 2007) and Glass et al (The Journal of Immunology, 2004), which are incorporated by reference herein in their entirety. Examples of antibodies that target TNFα include those described by Chatterjea et al (F1000Res, 2013) and Williams et al (PNAS, 1992), which are incorporated herein in their entirety. Examples of antibodies that target BMP2 include those described by Su et al (JBC, 2007), which is incorporated herein in its entirety.
[0059] One skilled in the art can determine the therapeutically effective amount of composition to administer to a site in need in an individual. Determination of efficacy can be determined by monitoring the site that has been administered the composition, such as by X-ray imaging or a physical examination. In particular embodiments, the amount of composition used may be approximately at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 ng either in total or per kilogram of the individual to be administered. In specific embodiments, the amount of composition used may be approximately at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 mg either in total or per kilogram of the individual to be administered. In some embodiments, the amount of composition used may be approximately at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mM of agent to be administered to the individual. In some embodiments, the administered concentration is or is about 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 110, 120, 125, 130, 140, or 150 ng/ml. Ranges may be or may be about 10-150, 10-140, 10-130, 10-125, 10-120, 10-110, 10-100, 10-90, 10-80, 10-75, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-150, 20-140, 20-130, 20-130, 20-125, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-150, 30-140, 30-130, 30-125, 30-120, 30-110, 30-100, 30-90, 30-80, 30-75, 30-70, 30-60, 30-50, 30-40, 40-150, 40-140-, 40-130, 40-125, 40-120, 40-110, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-150, 50-140, 50-130, 50-125, 50-120, 50-110, 50-100, 50-90, 50-80, 50-70, 50-60, 60-150, 60-140, 60-130, 60-125, 60-120, 60-110, 60-100, 60-90, 60-80, 60-75, 60-70, 70-150, 70-140, 70-130, 70-125, 70-120, 70-110, 70-100, 70-90, 70-80, 70-75, 80-150, 80-140, 80-130, 80-120, 80-110, 80-100, 80-90, 90-150, 90-140, 90-130, 90-125, 90-120, 90-110, 90-100, 100-150, 100-140, 100-130, 100-125, 100-120, 100-110, 110-150, 110-140, 110-130, 110-125, 110-120, 120-150, 120-140, 120-130, 120-125, 130-150, 130-140, or 140-150 ng/ml.
[0060] The number of administrations may be determined by one skilled in the art. In some embodiments, a single administration may be used, while in further embodiments, multiple administrations are given to the individual.
IV. Biocompatible Gel and/or Scaffold
[0061] In particular embodiments, the composition administered to the site in need may comprise a biocompatible gel and/or scaffold. Any biocompatible gel and/or scaffold may be used, wherein the gel and/or scaffold may enhance the efficacy of the cytokine or agent described above. The gel and/or scaffold may be comprised of matrigel, hydrogel, periosteum, hydroxyapatite, tricalcium phosphate, polystyrene, poly-1-lactic acid, polyglycolic acid, poly-dl-lactic-co-glycolic acid, collagen, proteoglycans, alginate-based substrates, chitosan, collagen-GAG, extracellular matrix, or a combination thereof. The gel and/or scaffold may be produced using any method known in the art. Gels and/or scaffolds that require being produced from an in vivo source, such as periosteum, may be from a source autologous, allogeneic, syngeneic, xenogeneic, or a combination thereof to the individual to be administered the composition at the site in need.
[0062] In particular embodiments, the cytokine(s) and/or agent(s) is/are combined with the biocompatible gel and/or scaffold in order to produce a new composition. The new composition may be used in the methods of the present disclosure. For example, CCL5 may be combined with matrigel to form a new composition that may be administered to a site in need, such as a bone break, in order to recruit P-SSCs to differentiate into osteoblasts and induce bone healing, while reducing the recruitment of OCPs, and thus reduce osteoclasts recruitment to the site in need.
V. Pharmaceutical Preparations
[0063] Pharmaceutical compositions of the present disclosure comprise an effective amount of one or more cytokines, CCR5 inhibitors, TNFα inhibitors, stem cells or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of an pharmaceutical composition that contains at least one cytokine, CCR5 inhibitor, TNFα inhibitor, or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21.sup.st Ed. Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
[0064] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
[0065] The cytokine or CCR5 inhibitor or TNFα inhibitor may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).
[0066] The cytokine or CCR5 inhibitor or TNFα inhibitor may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.
[0067] Further in accordance with the present disclosure, the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
[0068] In accordance with the present disclosure, the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.
[0069] In a specific embodiment of the present disclosure, the composition is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner such as grinding. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
[0070] In further embodiments, the present disclosure may concern the use of a pharmaceutical lipid vehicle compositions that include at least one cytokine or CCR5 inhibitor or TNFα inhibitor, one or more lipids, and an aqueous solvent. As used herein, the term “lipid” will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term “lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods of the present disclosure.
[0071] One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the cytokine or CCR5 inhibitor or TNFα inhibitor may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.
[0072] The actual dosage amount of a composition of the present disclosure administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
[0073] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
[0074] In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.
[0075] A. Alimentary Compositions and Formulations
[0076] In preferred embodiments of the present disclosure, the cytokine or CCR5 inhibitor or TNFα inhibitor are formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
[0077] In certain embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792, 451, each specifically incorporated herein by reference in its entirety). The tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Upon reaching the small intestines, the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells. A syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.
[0078] For oral administration the compositions of the present disclosure may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation. For example, a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively, the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
[0079] Additional formulations hat are suitable for other modes of alimentary administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
[0080] B. Parenteral Compositions and Formulations
[0081] In further embodiments, the cytokine or CCR5 inhibitor or TNFα inhibitor may be administered via a parenteral route. As used herein, the term “parenteral” includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).
[0082] Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
[0083] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
[0084] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.
[0085] C. Miscellaneous Pharmaceutical Compositions and Formulations
[0086] In other preferred embodiments of the invention, the active compound, comprising a cytokine or CCR5 inhibitor or TNFα inhibitor may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation.
[0087] Pharmaceutical compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder. Ointments include all oleaginous, adsorption, emulsion and water-soluble based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture. Transdermal administration of the present invention may also comprise the use of a “patch”. For example, the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.
[0088] In certain embodiments, the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).
[0089] The term aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms.
VI. Kits
[0090] Any of the compositions described herein may be comprised in a kit. In a non-limiting example, a cytokine or CCR5 inhibitor or TNFα inhibitor, lipid, and/or additional agent, may be comprised in a kit. The kits will thus comprise, in suitable container means, a cytokine or CCR5 inhibitor or TNFα inhibitor and a lipid, and/or an additional agent of the present disclosure.
[0091] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the cytokine or CCR5 inhibitor or TNFα inhibitor, lipid, additional agent, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
[0092] Therapeutic kits of the present disclosure are kits comprising a cytokine protein, polypeptide, peptide, inhibitor, gene, vector and/or other CCR5 effector. Such kits will generally contain, in suitable container means, a pharmaceutically acceptable formulation a cytokine protein, polypeptide, peptide, domain, inhibitor, and/or a gene and/or vector expressing any of the foregoing in a pharmaceutically acceptable formulation. The kit may have a single container means, and/or it may have distinct container means for each compound.
[0093] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The cytokine or CCR5 inhibitor or TNFα inhibitor compositions may also be formulated into a syringeable composition. In which case, the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit.
[0094] However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
[0095] The container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the cytokine protein, gene and/or inhibitory or CCR5 inhibitor formulation are placed, preferably, suitably allocated. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
[0096] The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained.
[0097] Irrespective of the number and/or type of containers, the kits of the invention may also comprise, and/or be packaged with, an instrument for assisting with the injection/administration and/or placement of the ultimate cytokine protein and/or gene composition or CCR5 inhibitor within the body of an animal. Such an instrument may be a syringe, pipette, forceps, and/or any such medically approved delivery vehicle.
VII. Examples
[0098] The following examples are included to demonstrate particular embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute particular modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
Example 1
The Mx1 and ASMA Combination Selectively Labels Endogenous P-SSCs
[0099] The origin of new osteoblasts, particularly at fracture sites, has long been an important question for biologists (Schindeler et al., 2008). Recent studies revealed that a subset of periosteal cells has skeletal progenitor function and plays an important role in bone repair (van Gastel et al., 2012). However, there is no known P-SSC specific marker, making the in vivo origin and regulatory mechanism of these cells elusive. Studies have reported that Mx1 labels endogenous osteogenic stem/progenitor cells that can maintain the majority (>80%) of the mature osteoblast population over time in the adult mouse (Park et al., 2012a). It was also found that these cells are present in the periosteum and supply new osteoblasts for bone healing. Recently, several models have been developed to label P-SSCs; however, one limitation of these models, like the Mx1 model, is that both P-SSCs and multiple cell types are labeled. Therefore, to further define a model enabling selective labeling of P-SSCs, Mx1-Cre.sup.+Rosa26-Tomato.sup.+ mice were crossed with other mesenchymal stem cell (MSC) reporter mouse lines, including αSMA-GFP (Grcevic et al., 2012), Nestin-GFP (Mendez-Ferrer et al., 2010), and CXCL12-GFP (Omatsu et al., 2010). Among the tested combinations, immunohistochemistry of trigenic Mx1-Cre.sup.+Rosa26-Tomato.sup.±αSMA-GFP.sup.+ (Mx1/Tomato/αSMA-GFP) mice after a wild-type BM transplant showed that the Mx1 and αSMA-GFP combination selectively labels a subset of periosteal cells (
[0100] Given that periosteal progenitor cells are reported to give rise to bone and cartilage in response to injury (Colnot, 2009), It was examined whether Mx1.sup.+αSMA.sup.+ periosteal cells contribute to the formation of osteoblasts during injury repair in vivo. To define the origin and dynamics of the osteoblasts located at injury sites, drill-hole defects (˜1 mm diameter) were generated in the tibia, using a needle (20 G), in Mx1/Tomato/αSMA-GFP dual reporter mice. Histology of tibia injury at day 14 revealed that Mx1.sup.+αSMA.sup.+ periosteal cells repopulate the outer layer of the callus (
Example 2
Mx1+P-SSCs are the Major Source of New Osteoblasts in Bone Healing In Vivo
[0101] It is possible that the periosteum contains Mx1.sup.− P-SSCs that can contribute to new osteoblasts at injury sites. Therefore, to distinguish between P-SSCs and mature osteoblasts in the periosteum and to improve specific cell quantitation during bone healing, trigenic Mx1-Cre.sup.+Rosa26-Tomato.sup.+ osteocalcin-GFP.sup.+ (Mx1/Tomato/Ocn-GFP) reporter mice were generated. In this model, when Mx1+P-SSCs (Tomato+) differentiate into mature Ocn-GFP.sup.+ osteoblasts they express Tomato and GFP, while mature osteoblasts from Mx1.sup.− P-SSCs express GFP alone (Park et al., 2014). In vivo imaging and immunohistochemistry analysis of Mx1/Tomato/Ocn-GFP femurs and tibias revealed that the induction of Mx1-Cre activity can label distinct periosteal stem/progenitor cells without detectable Ocn-GFP expression in the metaphysis (50±4%) and diaphysis (64±6%) (
[0102] To test whether Mx1.sup.+ P-SSCs are the main source of new osteoblasts during bone repair in vivo, drill-hole defects (˜1 mm diameter removal of bone and periosteum) were generated in the tibial diaphysis of Mx1/Tomato/Ocn-GFP mice. A drill-hole defect can potentially be a model of bone healing without compromising tissue architecture (endosteum vs periosteum) and achieve better quantification of stem cell responses (He et al., 2011). Immunohistochemistry analysis of the bone defect revealed that Mx1.sup.+ P-SSCs reconstitute the majority (˜80%) of outer callus-forming cells with new periosteum (Tomato+), and >70% of the bone-forming osteoblasts (Tomato.sup.+GFP.sup.+) surrounding new bone bridges (
Example 3
Mx1.SUP.+.αSMA.SUP.+ P-SSCs are Long-Term Repopulating Progenitors Required for Bone Healing
[0103] SSCs are known to be present in the BM (Worthley et al., 2015) and may contribute to bone healing if the periosteum is defective or removed. To determine if Mx1.sup.+ periosteal cells are essential for bone healing, an inducible deletion model was developed by intercrossing Mx1-Cre/Rosa26-Tomato mice with Rosa26-DTR mice (Park et al., 2012a) and the requirement for Mx1.sup.+ periosteal cells in bone healing was tested. To restrict the conditional deletion of Mx1.sup.+ cells to the periosteum, local surface administration of diphtheria toxin (DT) (20 μL, 1 μg/mL) was used to cover an area with ˜1 cm of diameter at the junction of the sagittal and coronal sutures (
[0104] An important characteristic of stem cells is their long-term repopulation capability after multiple rounds of injuries (Sacchetti et al., 2007). Given that a subset of Mx1.sup.+αSMA.sup.+ periosteal cells remains on the outer layer of the callus even after bone healing, the stem cell function of these cells was tested by examining the long-term repopulation ability of Mx1.sup.+αSMA.sup.+ periosteal cells and their contribution to bone repair after several rounds of injuries. Consecutive intravital imaging of the primary injury sites of Mx1/Tomato/αSMA-GFP mice at ˜4 months of age (
[0105] To better define the long-term repopulation characteristics and to exclude the possibility that Mx1.sup.+αSMA.sup.− periosteal cells include P-SSCs and become Mx1.sup.+αSMA.sup.+ cells in bone injury, we performed serial transplantations of these cells (
Example 4
Mx1.SUP.+ Periosteal Cells, Distinct from Nestin-GFP+BM-SSCs, Supply New Osteoblasts in Fracture Healing
[0106] BM-SSCs were reported to localize in perivascular regions with Nestin-GFP expression (Mendez-Ferrer et al.). In addition, the CD31.sup.−Nestin-GFP.sup.+ perivascular reticular cells are LepR-Cre descendants and contribute to postnatal bone regeneration (Kunisaki et al., 2013). Mx1.sup.+ cells have been shown to overlap with a subset of Nestin.sup.+ populations in the BM (Park et al., 2012a), and therefore researchers developed the trigenic Mx1-Cre.sup.+Rosa26-Tomato.sup.+Nestin-GFP+(Mx1/Tomato/Nestin-GFP) dual reporter mouse and tested if Mx1 and Nestin double labeling can distinguish BM-SSCs from P-SSCs and their contributions to osteolineage cells in bone injury. In vivo imaging and FACS analysis of these mice revealed that there are two distinct cell populations (Mx1.sup.+Nestin.sup.+; ˜27% and Mx1.sup.−Nestin.sup.+; ˜73%) within Nestin.sup.+ perivascular cells, whereas Mx1.sup.+ periosteal cells are exclusively Nestin negative (
Example 5
Mx1.SUP.+.aSMA.SUP.+ P-SSCs have a Unique Migratory Mechanism Regulated by Ccr5 In Vivo
[0107] Skeletal progenitors have been proposed to have migratory or circulatory potential. However, there is no in vivo evidence for the endogenous migratory mechanism of P-SSCs. In particular, how P-SSCs are signaled to respond to injury and how they migrate and form fracture-repairing osteoblasts is essentially unknown. To test whether P-SSCs migrate toward the injury site in vivo, continuous Z-stack imaging of individual Mx1.sup.+αSMA.sup.+ P-SSCs was performed and sequentially scanning was performed on the detailed structures of the periosteum, bone matrix, and BM near the injury sites immediately (0), 24, and 48 hours after injury (
[0108] Many growth factors and cytokines are known to enhance the migration and proliferation of in vitro-expanded MSCs (Einhorn and Gerstenfeld, 2015; Schindeler et al., 2008). However, there is no direct evidence of the effect of these molecules on the in vivo migration potential of endogenous SSCs. molecules involved in P-SSC migration were examined by Affymetrix-based global gene expression analysis of Mx1.sup.+Ocn.sup.− periosteal cells compared to more differentiated cells and Nestin.sup.+ BM cells (
[0109] It may be possible that Mx1 does not label all SSCs in the BM and that Mx1.sup.− BM-SSCs can express CCR3/5. Using LepR-Cre.sup.+Rosa26-Tomato.sup.+ reporter mice, generated to label postnatal BM-SSCs that are the major source of new osteoblasts and adipocytes in the BM (Zhou et al., 2014), LepR.sup.+ cells were tested in the BM for expression of CCR5 and their contribution to osteolineage cells in injury repair. In vivo imaging and FACS analysis of these mice revealed that the LepR.sup.+ cells are present in both the periosteum and the BM (
[0110] A previous study reported that Prx1-GFP.sup.+ cells in the periosteum have progenitor characteristics and contribute to injury repair (Ouyang et al., 2013). Therefore, to exclude any possible contamination of CCR5.sup.+ hematopoietic cells in the Mx1.sup.+αSMA.sup.+ P-SSC fraction, FACS analysis of periosteal cells from Prx1-CreER-GFP mice was performed and it was confirmed that Prx1-GFP.sup.+ P-SSCs (CD45.sup.−CD31.sup.−TER119.sup.−CD140a.sup.+GFP.sup.+) have high expression of CCR5 (86%) on the cell surface (
Example 6
CCL5 Induces the In Vivo Migration of P-SSCs
[0111] In vitro SSC migration followed by four-day colony formation assays with various bone growth factors revealed that tumor necrosis factor alpha (TNFα) and CCL5 (a common ligand for CCR3 and CCR5) (Blanpain et al., 2001) significantly enhance the migration capability of Mx1+P-SSCs (
Example 7
CCL5 is Useful for Bone Healing, and Local Provision of CCL5 Accelerates Healing of Aged-Bone Defects with Increased P-SSC Recruitment
[0112] The development of novel therapeutic avenues to accelerate early bone healing will eventually benefit patients with devastating skeletal injuries. Given the CCL5-mediated migration of Mx1.sup.+αSMA.sup.+ P-SSCs and their recruitment toward injury sites, the in vivo contribution of CCL5 to the repair of fractured bones was assessed in an animal model. To test the physiologic importance of CCL5 in P-SSC migration and in injury repair, the bone healing of Ccl5-deficient mice was compared to that of WT litter mates. While there were no noticeable differences in bone parameters (BV/TV, travecular numbers and trabecular thickness) of 3-month-old Ccl5-deficient mice (data not shown), external callus mineralization (External BV/TV) and new bone formation (BV/TV) at defect sites 10 days after injury were significantly reduced (˜35%, p<0.01) in Ccl5-deficient mice compared to WT controls (
[0113] Bone injuries were introduced to mouse calvaria of Mx1/Tomato/αSMA-GFP mice. Local treatment with CCL5 (+CCL5) or CXCL12 (+CXCL12) mixed with Matrigel (2 μL at 10 ng/μL) or Matrigel alone (CON) was given at the site of mechanical injury on days 0, 2, and 4. Treatment with CCL5 induced rapid recruitment of Mx1.sup.+αSMA.sup.+ P-SSCs at day 5 and substantially increased their number at day 10 with accelerated bone mineralization (
Example 8
The Deletion and Inhibition of CCR5 Delay P-SSC Migration and Bone Repair In Vivo
[0114] During live imaging of Mx1/Tom/SMA reporter mice treated with a pharmacological CCR5 inhibitor (
Example 9
In Vivo Imaging of Osteoclastic Response to Ccl5 or Inhibition of Ccr5
[0115] The assessment of the in vivo contribution of CCL5 or CCR5 inhibitor (maraviroc) treatments on osteoclast function in relation to bone healing was performed. An animal model was developed in which it is possible to track the osteoclast response to bone injury. CathepsinK (Ctsk) is a known marker of post-natal osteoclasts. Developmentally both mesenchymal and hematopoietic cell linages are Tom+ in CtskCre/Tom reporter mice, which is evident by the imbedding of Tom+ cells within the bone and abundance of Tom+ cells within the bone marrow. To achieve selective labeling of hematopoietic linage osteoclast, a bone marrow transplant into wild type (WT) recipient mice is necessary. Seven-week-old WT mice were lethally irradiated with 9.50 Gy and the following day 10 million CtskCre/Tom bone marrow cells were transplanted (CtskCre-BM). The bone marrow cells were allowed to repopulate for at least 6 weeks prior to experimentation. To test if stimulation or inhibition of CCR5 prevents or induces osteoclast differentiation and/or migration in response to an injury, in vivo intravital imaging of Ctsk.sup.+ cell response to a calvarial injury was performed.
[0116] An injury was induced with a 27-gauge needle and mice received one of 3 treatments at the injury sight: 1) control (2 uL Matrigel) Day 0, 2, and 4; 2) CCL5 (2 uL, 10 ng/uL in Matrigel) Day 0, 2, and 4; or 3) a CCR5 inhibitor (10 uL, 4.9 mM Maraviroc in Matrigel) on Day 0, 2, 4, 7, and 10 post-injury (
Example 10
Injury Repair in CCL5.SUP.−/− Mice
[0117] Based off findings that CCL5 treatment to bone injuries significantly increased the number of P-SSCs recruited to the injury sites and accelerates bone healing, the inventors next sought to determine if loss of CCL5 has deleterious effects on bone injury repair (see
[0118] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the design as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the present disclosure, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.
TABLE-US-00001 SEQUENCES SEQ ID NO: 1: 1 gctgcagagg attcctgcag aggatcaaga cagcacgtgg acctcgcaca gcctctccca 61 caggtaccat gaaggtctcc gcggcagccc tcgctgtcat cctcattgct actgccctct 121 gcgctcctgc atctgcctcc ccatattcct cggacaccac accctgctgc tttgcctaca 181 ttgcccgccc actgccccgt gcccacatca aggagtattt ctacaccagt ggcaagtgct 241 ccaacccagc agtcgtccac aggtcaagga tgccaaagag agagggacag caagtctggc 301 aggatttcct gtatgactcc cggctgaaca agggcaagct ttgtcacccg aaagaaccgc 361 caagtgtgtg ccaacccaga gaagaaatgg gttcgggagt acatcaactc tttggagatg 421 agctaggatg gagagtcctt gaacctgaac ttacacaaat ttgcctgttt ctgcttgctc 481 ttgtcctagc ttgggaggct tcccctcact atcctacccc acccgctcct tgaagggccc 541 agattctacc acacagcagc agttacaaaa accttcccca ggctggacgt ggtggctcac 601 gcctgtaatc ccagcacttt gggaggccaa ggtgggtgga tcacttgagg tcaggagttc 661 gagaccagcc tggccaacat gatgaaaccc catctctact aaaaatacaa aaaattagcc 721 gggcgtggta gcgggcgcct gtagtcccag ctactcggga ggctgaggca ggagaatggc 781 gtgaacccgg gaggcggagc ttgcagtgag ccgagatcgc gccactgcac tccagcctgg 841 gcgacagagc gagactccgt ctcaaaaaaa aaaaaaaaaa aaaaaataca aaaattagcc 901 gggcgtggtg gcccacgcct gtaatcccag ctactcggga ggctaaggca ggaaaattgt 961 ttgaacccag gaggtggagg ctgcagtgag ctgagattgt gccacttcac tccagcctgg 1021 gtgacaaagt gagactccgt cacaacaaca acaacaaaaa gcttccccaa ctaaagccta 1081 gaagagcttc tgaggcgctg ctttgtcaaa aggaagtctc taggttctga gctctggctt 1141 tgccttggct ttgccagggc tctgtgacca ggaaggaagt cagcatgcct ctagaggcaa 1201 ggaggggagg aacactgcac tcttaagctt ccgccgtctc aacccctcac aggagcttac 1261 tggcaaacat gaaaaatcgg cttaccatta aagttctcaa tgcaaccata aaaaaaaaa SEQ ID NO: 2: 1 gctgcagagg attcctgcag aggatcaaga cagcacgtgg acctcgcaca gcctctccca 61 caggtaccat gaaggtctcc gcggcagccc tcgctgtcat cctcattgct actgccctct 121 gcgctcctgc atctgcctcc ccatattcct cggacaccac accctgctgc tttgcctaca 181 ttgcccgccc actgccccgt gcccacatca aggagtattt ctacaccagt ggcaagtgct 241 ccaacccagc agtcgtcttt gtcacccgaa agaaccgcca agtgtgtgcc aacccagaga 301 agaaatgggt tcgggagtac atcaactctt tggagatgag ctaggatgga gagtccttga 361 acctgaactt acacaaattt gcctgtttct gcttgctctt gtcctagctt gggaggcttc 421 ccctcactat cctaccccac ccgctccttg aagggcccag attctaccac acagcagcag 481 ttacaaaaac cttccccagg ctggacgtgg tggctcacgc ctgtaatccc agcactttgg 541 gaggccaagg tgggtggatc acttgaggtc aggagttcga gaccagcctg gccaacatga 601 tgaaacccca tctctactaa aaatacaaaa aattagccgg gcgtggtagc gggcgcctgt 661 agtcccagct actcgggagg ctgaggcagg agaatggcgt gaacccggga ggcggagctt 721 gcagtgagcc gagatcgcgc cactgcactc cagcctgggc gacagagcga gactccgtct 781 caaaaaaaaa aaaaaaaaaa aaaatacaaa aattagccgg gcgtggtggc ccacgcctgt 841 aatcccagct actcgggagg ctaaggcagg aaaattgttt gaacccagga ggtggaggct 901 gcagtgagct gagattgtgc cacttcactc cagcctgggt gacaaagtga gactccgtca 961 caacaacaac aacaaaaagc ttccccaact aaagcctaga agagcttctg aggcgctgct 1021 ttgtcaaaag gaagtctcta ggttctgagc tctggctttg ccttggcttt gccagggctc 1081 tgtgaccagg aaggaagtca gcatgcctct agaggcaagg aggggaggaa cactgcactc 1141 ttaagcttcc gccgtctcaa cccctcacag gagcttactg gcaaacatga aaaatcggct 1201 taccattaaa gttctcaatg caaccataaa aaaaaaa SEQ ID NO: 3 1 mkvsaaalav iliatalcap asaspyssdt tpccfayiar plprahikey fytsgkcsnp 61 avvhrsrmpk regqqvwqdf lydsrlnkgk lchpkeppsv cqpreemgsg vhqlfgdelg 121 wrvlepeltq iclfllalvl aweasphypt ppap SEQ ID NO: 4 1 mkvsaaalav iliatalcap asaspyssdt tpccfayiar plprahikey fytsgkcsnp 61 avvfvtrknr qvcanpekkw vreyinslem s SEQ ID NO: 5 1 cagacgctcc ctcagcaagg acagcagagg accagctaag agggagagaa gcaactacag 61 accccccctg aaaacaaccc tcagacgcca catcccctga caagctgcca ggcaggttct 121 cttcctctca catactgacc cacggctcca ccctctctcc cctggaaagg acaccatgag 181 cactgaaagc atgatccggg acgtggagct ggccgaggag gcgctcccca agaagacagg 241 ggggccccag ggctccaggc ggtgcttgtt cctcagcctc ttctccttcc tgatcgtggc 301 aggcgccacc acgctcttct gcctgctgca ctttggagtg atcggccccc agagggaaga 361 gttccccagg gacctctctc taatcagccc tctggcccag gcagtcagat catcttctcg 421 aaccccgagt gacaagcctg tagcccatgt tgtagcaaac cctcaagctg aggggcagct 481 ccagtggctg aaccgccggg ccaatgccct cctggccaat ggcgtggagc tgagagataa 541 ccagctggtg gtgccatcag agggcctgta cctcatctac tcccaggtcc tcttcaaggg 601 ccaaggctgc ccctccaccc atgtgctcct cacccacacc atcagccgca tcgccgtctc 661 ctaccagacc aaggtcaacc tcctctctgc catcaagagc ccctgccaga gggagacccc 721 agagggggct gaggccaagc cctggtatga gcccatctat ctgggagggg tcttccagct 781 ggagaagggt gaccgactca gcgctgagat caatcggccc gactatctcg actttgccga 841 gtctgggcag gtctactttg ggatcattgc cctgtgagga ggacgaacat ccaaccttcc 901 caaacgcctc ccctgcccca atccctttat taccccctcc ttcagacacc ctcaacctct 961 tctggctcaa aaagagaatt gggggcttag ggtcggaacc caagcttaga actttaagca 1021 acaagaccac cacttcgaaa cctgggattc aggaatgtgt ggcctgcaca gtgaagtgct 1081 ggcaaccact aagaattcaa actggggcct ccagaactca ctggggccta cagctttgat 1141 ccctgacatc tggaatctgg agaccaggga gcctttggtt ctggccagaa tgctgcagga 1201 cttgagaaga cctcacctag aaattgacac aagtggacct taggccttcc tctctccaga 1261 tgtttccaga cttccttgag acacggagcc cagccctccc catggagcca gctccctcta 1321 tttatgtttg cacttgtgat tatttattat ttatttatta tttatttatt tacagatgaa 1381 tgtatttatt tgggagaccg gggtatcctg ggggacccaa tgtaggagct gccttggctc 1441 agacatgttt tccgtgaaaa cggagctgaa caataggctg ttcccatgta gccccctggc 1501 ctctgtgcct tcttttgatt atgtttttta aaatatttat ctgattaagt tgtctaaaca 1561 atgctgattt ggtgaccaac tgtcactcat tgctgagcct ctgctcccca ggggagttgt 1621 gtctgtaatc gccctactat tcagtggcga gaaataaagt ttgcttagaa aagaaaaaaa 1681 aaaaaa SEQ ID NO: 6 1 cagacgctcc ctcagcaagg acagcagagg accagctaag agggagagaa gcaactacag 61 accccccctg aaaacaaccc tcagacgcca catcccctga caagctgcca ggcaggttct 121 cttcctctca catactgacc cacggctcca ccctctctcc cctggaaagg acaccatgag 181 cactgaaagc atgatccggg acgtggagct ggccgaggag gcgctcccca agaagacagg 241 ggggccccag ggctccaggc ggtgcttgtt cctcagcctc ttctccttcc tgatcgtggc 301 aggcgccacc acgctcttct gcctgctgca ctttggagtg atcggccccc agagggaaga 361 gttccccagg gacctctctc taatcagccc tctggcccag gcagtcagat catcttctcg 421 aaccccgagt gacaagcctg tagcccatgt tgtagcaaac cctcaagctg aggggcagct 481 ccagtggctg aaccgccggg ccaatgccct cctggccaat ggcgtggagc tgagagataa 541 ccagctggtg gtgccatcag agggcctgta cctcatctac tcccaggtcc tcttcaaggg 601 ccaaggctgc ccctccaccc atgtgctcct cacccacacc atcagccgca tcgccgtctc 661 ctaccagacc aaggtcaacc tcctctctgc catcaagagc ccctgccaga gggagacccc 721 agagggggct gaggccaagc cctggtatga gcccatctat ctgggagggg tcttccagct 781 ggagaagggt gaccgactca gcgctgagat caatcggccc gactatctcg actttgccga 841 gtctgggcag gtctactttg ggatcattgc cctgtgagga ggacgaacat ccaaccttcc 901 caaacgcctc ccctgcccca atccctttat taccccctcc ttcagacacc ctcaacctct 961 tctggctcaa aaagagaatt gggggcttag ggtcggaacc caagcttaga actttaagca 1021 acaagaccac cacttcgaaa cctgggattc aggaatgtgt ggcctgcaca gtgaagtgct 1081 ggcaaccact aagaattcaa actggggcct ccagaactca ctggggccta cagctttgat 1141 ccctgacatc tggaatctgg agaccaggga gcctttggtt ctggccagaa tgctgcagga 1201 cttgagaaga cctcacctag aaattgacac aagtggacct taggccttcc tctctccaga 1261 tgtttccaga cttccttgag acacggagcc cagccctccc catggagcca gctccctcta 1321 tttatgtttg cacttgtgat tatttattat ttatttatta tttatttatt tacagatgaa 1381 tgtatttatt tgggagaccg gggtatcctg ggggacccaa tgtaggagct gccttggctc 1441 agacatgttt tccgtgaaaa cggagctgaa caataggctg ttcccatgta gccccctggc 1501 ctctgtgcct tcttttgatt atgtttttta aaatatttat ctgattaagt tgtctaaaca 1561 atgctgattt ggtgaccaac tgtcactcat tgctgagcct ctgctcccca ggggagttgt 1621 gtctgtaatc gccctactat tcagtggcga gaaataaagt ttgcttagaa aagaaaaaaa 1681 aaaaaa SEQ ID NO: 7 1 ccacaaaggg cacttggccc cagggctagg agagcgaggg gagagcacag ccacccgcct 61 cggcggcccg ggactcggct cgactcgccg gagaatgcgc ccgaggacga cggggcgcca 121 gagccgcggt gctttcaact ggcgagcgcg aatgggggtg cactggagta aggcagagtg 181 atgcgggggg gcaactcgcc tggcaccgag atcgccgccg tgcccttccc tggacccggc 241 gtcgcccagg atggctgccc cgagccatgg gccgcggcgg agctagcgcg gagcgcccga 301 ccctcgaccc ccgagtcccg gagccggccc cgcgcggggc cacgcgtccc tcgggcgctg 361 gttcctaagg aggacgacag caccagcttc tcctttctcc cttcccttcc ctgccccgca 421 ctcctccccc tgctcgctgt tgttgtgtgt cagcacttgg ctggggactt cttgaacttg 481 cagggagaat aacttgcgca ccccactttg cgccggtgcc tttgccccag cggagcctgc 541 ttcgccatct ccgagcccca ccgcccctcc actcctcggc cttgcccgac actgagacgc 601 tgttcccagc gtgaaaagag agactgcgcg gccggcaccc gggagaagga ggaggcaaag 661 aaaaggaacg gacattcggt ccttgcgcca ggtcctttga ccagagtttt tccatgtgga 721 cgctctttca atggacgtgt ccccgcgtgc ttcttagacg gactgcggtc tcctaaaggt 781 cgaccatggt ggccgggacc cgctgtcttc tagcgttgct gcttccccag gtcctcctgg 841 gcggcgcggc tggcctcgtt ccggagctgg gccgcaggaa gttcgcggcg gcgtcgtcgg 901 gccgcccctc atcccagccc tctgacgagg tcctgagcga gttcgagttg cggctgctca 961 gcatgttcgg cctgaaacag agacccaccc ccagcaggga cgccgtggtg cccccctaca 1021 tgctagacct gtatcgcagg cactcaggtc agccgggctc acccgcccca gaccaccggt 1081 tggagagggc agccagccga gccaacactg tgcgcagctt ccaccatgaa gaatctttgg 1141 aagaactacc agaaacgagt gggaaaacaa cccggagatt cttctttaat ttaagttcta 1201 tccccacgga ggagtttatc acctcagcag agcttcaggt tttccgagaa cagatgcaag 1261 atgctttagg aaacaatagc agtttccatc accgaattaa tatttatgaa atcataaaac 1321 ctgcaacagc caactcgaaa ttccccgtga ccagactttt ggacaccagg ttggtgaatc 1381 agaatgcaag caggtgggaa agttttgatg tcacccccgc tgtgatgcgg tggactgcac 1441 agggacacgc caaccatgga ttcgtggtgg aagtggccca cttggaggag aaacaaggtg 1501 tctccaagag acatgttagg ataagcaggt ctttgcacca agatgaacac agctggtcac 1561 agataaggcc attgctagta acttttggcc atgatggaaa agggcatcct ctccacaaaa 1621 gagaaaaacg tcaagccaaa cacaaacagc ggaaacgcct taagtccagc tgtaagagac 1681 accctttgta cgtggacttc agtgacgtgg ggtggaatga ctggattgtg gctcccccgg 1741 ggtatcacgc cttttactgc cacggagaat gcccttttcc tctggctgat catctgaact 1801 ccactaatca tgccattgtt cagacgttgg tcaactctgt taactctaag attcctaagg 1861 catgctgtgt cccgacagaa ctcagtgcta tctcgatgct gtaccttgac gagaatgaaa 1921 aggttgtatt aaagaactat caggacatgg ttgtggaggg ttgtgggtgt cgctagtaca 1981 gcaaaattaa atacataaat atatatatat atatatattt tagaaaaaag aaaaaaacaa 2041 acaaacaaaa aaaccccacc ccagttgaca ctttaatatt tcccaatgaa gactttattt 2101 atggaatgga atggaaaaaa aaacagctat tttgaaaata tatttatatc tacgaaaaga 2161 agttgggaaa acaaatattt taatcagaga attattcctt aaagatttaa aatgtattta 2221 gttgtacatt ttatatgggt tcaaccccag cacatgaagt ataatggtca gatttatttt 2281 gtatttattt actattataa ccacttttta ggaaaaaaat agctaatttg tatttatatg 2341 taatcaaaag aagtatcggg tttgtacata attttccaaa aattgtagtt gttttcagtt 2401 gtgtgtattt aagatgaaaa gtctacatgg aaggttactc tggcaaagtg cttagcacgt 2461 ttgctttttt gcagtgctac tgttgagttc acaagttcaa gtccagaaaa aaaaagtgga 2521 taatccactc tgctgacttt caagattatt atattattca attctcagga atgttgcaga 2581 gtgattgtcc aatccatgag aatttacatc cttattaggt ggaatatttg gataagaacc 2641 agacattgct gatctattat agaaactctc ctcctgcccc ttaatttaca gaaagaataa 2701 agcaggatcc atagaaataa ttaggaaaac gatgaacctg caggaaagtg aatgatggtt 2761 tgttgttctt ctttcctaaa ttagtgatcc cttcaaaggg gctgatctgg ccaaagtatt 2821 caataaaacg taagatttct tcattattga tattgtggtc atatatattt aaaattgata 2881 tctcgtggcc ctcatcaagg gttggaaatt tatttgtgtt ttacctttac ctcatctgag 2941 agctctttat tctccaaaga acccagtttt ctaacttttt gcccaacacg cagcaaaatt 3001 atgcacatcg tgttttctgc ccaccctctg ttctctgacc tatcagcttg cttttctttc 3061 caaggttgtg tgtttgaaca catttctcca aatgttaaac ctatttcaga taataaatat 3121 caaatctctg gcatttcatt ctataaagtc caacctgtaa gagaaaaaaa aaaaaaaaaa 3181 aaaaaaaaaa a SEQ ID NO: 8 1 mvagtrclla lllpqvllgg aaglvpelgr rkfaaassgr pssqpsdevl sefelrllsm 61 fglkqrptps rdavvppyml dlyrrhsgqp gspapdhrle raasrantvr sfhheeslee 121 lpetsgkttr rfffnlssip teefitsael qvfreqmqda lgnnssfhhr iniyeiikpa 181 tanskfpvtr lldtrlvnqn asrwesfdvt pavmrwtaqg hanhgfvvev ahleekqgvs 241 krhvrisrsl hqdehswsqi rpllvtfghd gkghplhkre krqakhkqrk rlkssckrhp 301 lyvdfsdvgw ndwivappgy hafychgecp fpladhlnst nhaivqtlvn svnskipkac 361 cvptelsais mlyldenekv vlknyqdmvv egcgcr