A motile injectable cell that accelerates musculoskeletal connective tissue repair

Abstract

Embodiments of the present invention disclose a genetically modified cell that accelerates tissue repair by overexpressing CXCR4. This motile cell can be administered to subjects in need, enhancing tissue regeneration compared to unmodified cells. The cell may include various progenitor cell lines and demonstrates low NF-Kb pathway activity, which suppresses catabolic activity and promotes anabolism in inflammatory environments. The invention also encompasses methods of treatment using these cells, compositions containing them, and kits for their delivery.

Description

Attorney Docket No.: 405002-554001WO A MOTILE INJECTABLE CELL THAT ACCELERATES MUSCULOSKELETAL CONNECTIVE TISSUE REPAIR CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to United States Provisional Patent No. 63 / 622,027, filed on 17-January-2024, the entirety of which is incorporated by reference as if fully reproduced and set forth herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under R21 AR077326 awarded by the National Institutes of Health. The government has certain rights in the invention. BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0003] This submission is accompanied by a “Sequence Listing XML” containing SEQ ID NOs: 1-2 and created on 16-January-2024, 4 KB (kilobytes) size, and submitted with the filename: “405002-554P01US.xml”. The “Sequence Listing XML” was submitted with United States Provisional Patent No.63 / 622,027 (referenced above). None of the sequences therein contain less than 10 amino acids in length or less than 10 nucleotides and thus none are mandatorily marked as intentionally skipped sequences under WIPO Sequence software version 2.3.0. The Sequence Listing XML was generated using WIPO Sequence software version 2.3.0, in accordance with 37 CFR §§ 1.831 through 1.835, and is herewith submitted as an XML file, via the USPTO patent electronic filing system, 37 CFR § 1.835(a)(1). The Sequence Listing XML file is incorporated by reference herein in its entirety. FIELD OF THE INVENTION

[0004] This application relates to specialized compositions, modified cells, and targeted methods for accelerating the healing of tissue injuries, for example, for proteoglycan containing connective tissues (e.g., cartilage, fibrocartilage) and related structures such as the menisci in articulations and joints. BACKGROUND OF THE INVENTION

[0005] Anterior cruciate ligament (ACL) injuries are the most common ligament injury in the United StatesA. Unfortunately, ACL injuries can be severely painful for all individuals and canAttorney Docket No.: 405002-554001WO typically include an acute knee meniscus injury (e.g., meniscus tear) or can lead to a chronic meniscus injury and complications over time, especially if surgical correction of the ACL damage is not performed promptly. In the knee, a meniscus injury is the second most common injury after ACLB. Meniscus surgery can be performed simultaneously with ACL surgery or separately.

[0006] Meniscus surgery most commonly includes a trimming of a torn area of the meniscus, a partial meniscectomy, or an attempt at reconstruction. A second type of repair is a suturing of the meniscus in attempts to hold a cracked or torn area together. Healing of the meniscus is extremely slow. Blood flow in the menisci decreases with age and the central areas (i.e., areas central to major joints) are avascular in adults, relying on diffusion to the central areas from the periphery, and some areas are incapable of healing. While healing can be halted or slow, the menisci must still act (or perform) to reduce friction on major joints and to support weight, so the injured subject is immobilized and usually in pain during the healing or rehabilitation process. While this leads to immobilization of athletes, it also leads to distress, lost days at work, discomfort, and significant costs to the economyC. While ACL surgeries provide a clear example of damage to the meniscus of the knee, menisci in other joints such as the wrist, acromioclavicular, sternoclavicular, and temporomandibular joints can be equally or more painful during injury and slow to heal. Additionally, the meniscus might not heal at all, and the injury can develop into debilitating osteoarthritis. As such, there is an ongoing need for new methods and approaches to accelerating the healing of injuries to injured tissues. BRIEF SUMMARY OF THE INVENTION

[0007] In summary embodiments for discussion, the technology herein includes a CXCR4 overexpressing cell that accelerates tissue repair in a subject by shortening the tissue repair time, and more particularly, to a genetically modified motile cell that enhances tissue repair efficiency. The technology disclosed herein can accelerate musculoskeletal connective tissue repairs and has many other benefits, as demonstrated in animal studies. For example, the acceleration of repairs can lower the risk of a development of post-traumatic osteoarthritis (PTOA) and other painful complications. Meanwhile, the technology disclosed herein can improve patient outcomes for old and young, improve the outlook for athletes after injuries, and lower the amount of time required for healing.

[0008] Tissue repair is a critical process in the human body, essential for restoring the function and structure of damaged tissues. This process can be slow and inefficient, particularly in cases of severe injury or in patients with compromised healing abilities. Traditional methods of enhancing tissue repair, such as surgical interventions and pharmacological treatments, oftenAttorney Docket No.: 405002-554001WO come with limitations and side effects. As a result, there is a growing interest in developing innovative approaches that can more effectively and safely accelerate tissue repair.

[0009] An example of our promising area of research involves the use of genetically modified cells to enhance the body's natural healing processes. By altering specific genetic pathways, we aim to create cells that can more efficiently migrate to injury sites, promote tissue regeneration, and reduce healing times. The chemokine receptor CXCR4 has been identified herein as a key player in cell migration and tissue repair, making it a target for genetic modification. Enhancing the expression of CXCR4 in motile cells could potentially improve their ability to reach and repair damaged tissues, offering a novel approach to accelerate healing in patients who require faster recovery. Thus, embodiments of the present invention disclose a cell (or a plurality of the cell line) that accelerates a musculoskeletal connective tissue repair, the cell including a genetic modification such that the cell is a CXCR4 overexpressing (CXCR4-OE) cell. According to some aspects, the cell is a motile cell that can be administered to a subject in need thereof; and the cell accelerates musculoskeletal connective tissue repair compared to the same cell line that is not carrying the genetic modification. In examples discussed herein, the cell comprises a cartilage-derived progenitor cell line (CPC).

[0010] In some embodiments, the cell disclosed above has a low NF-Kb pathway activity in response to SDF-1 (i.e., this means it has suppressed catabolic activity, which is important for cell-mediated anabolism especially in an inflammatory microenvironment, like the injured knee).

[0011] According to some aspects, a pharmaceutical formulation comprising any of the cells discussed above is provided. In some embodiments, a method of treating a subject diagnosed with a musculoskeletal connective tissue injury or a subject suspected of having a musculoskeletal connective tissue injury is disclosed herein, the method comprising the steps of: (1) obtaining a pharmaceutical formulation comprising a plurality of cells comprising a genetic modification such that the cells are CXCR4 overexpressing (CXCR4-OE); and (2) administering a therapeutically effective amount of the cells via administering the pharmaceutical formulation.

[0012] In this example of the method, a healing or repair of the musculoskeletal connective tissue injury is accelerated compared to a treating using the same formulation without the CXCR4- OE.

[0013] In the methods and the pharmaceutical formulations, in some embodiments, the pharmaceutical formulation is in the form of a suspension of the cells. The suspension can be configured for pharmaceutical administration by any method known in the arts.

[0014] According to some aspects, the connective tissue comprises a meniscus and / or a fibrocartilaginous tissue.Attorney Docket No.: 405002-554001WO

[0015] In the methods and formulations, according to some aspects, the cell comprises a cartilage-derived progenitor cell line (CPC).

[0016] In some embodiments, the administering to the subject is via an intra-articular injection at or near an area of injury.

[0017] In some embodiments, a kit for accelerating a musculoskeletal connective tissue repair in a subject in need thereof or in a subject suspected of having a musculoskeletal connective tissue injury is disclosed herein, the kit comprising: (1) a pharmaceutical formulation operative for accelerating a musculoskeletal connective tissue repair, the pharmaceutical formulation including: a CPC cell that is CXCR4-OE; and a solvent, powder, gas, or material that is capable of suspending the cell; and (2) a delivery device or configuration capable of delivering the suspended cell to a subject in need thereof.

[0018] In an invention brief summary or in a brief summary discussion list, the technology disclosed herein can be discussed, for example, in a summary discussion, by reviewing / discussing the following brief list of features, which can be inter-combined with any other embodiment, detail, aspect, or example disclosed herein:

[0019] Feature 1: A cell that accelerates musculoskeletal connective tissue repair, the cell comprising: a genetic modification such that the cell is a CXCR4 overexpressing (CXCR4 OE) cell; wherein the cell is a motile cell that can be administered to a subject in need thereof; and the cell accelerates musculoskeletal connective tissue repair compared to the same cell line that is not carrying the genetic modification.

[0020] Feature 2: The cell of feature 1, wherein the cell comprises any cell that comes from connective tissue, such as (but not limited to) a cartilage-derived progenitor cell line (CPC), a tenocyte, a chondrocyte, an adipocyte, an osteoblast, an osteocyte, an osteoclast, a synovial cell, a meniscal cell, and / or a mesenchymal stem cell.

[0021] Feature 3: The cell of feature 1 or feature 2, wherein the cell comprises a low NF- Kb pathway activity in response to SDF-1 providing a suppressed catabolic activity, which is operative to affect cell-mediated anabolism in an inflammatory microenvironment; and wherein a low NF-Kb pathway is such that the NF-Kb pathway of the cell is lower than that, all other things being equal, of a cell without the genetic modification of feature 1.

[0022] Feature 4: The cell of feature 1, wherein the cell is included in a pharmaceutically acceptable formulation.

[0023] Feature 5: The cell of feature 1, wherein the cell increases tissue regeneration in the subject compared to an unmodified cell counterpart.Attorney Docket No.: 405002-554001WO

[0024] Feature 6: A method of treating a subject diagnosed with a tissue injury or a subject suspected of having a tissue injury, the method comprising the steps of: (1) obtaining a pharmaceutical formulation comprising a plurality of cells comprising a genetic modification such that the cells are CXCR4 overexpressing (CXCR4-OE); and (2) administering a therapeutically effective amount of the cells via administering the pharmaceutical formulation; whereby a healing or repair of the tissue injury is accelerated compared to a treating using the same formulation without the CXCR4-OE.

[0025] Feature 7: The method of feature 6, wherein the cell comprises any cell that comes from connective tissue, such as (but not limited to) a cartilage-derived progenitor cell line (CPC), a tenocyte, a chondrocyte, an adipocyte, an osteoblast, an osteocyte, an osteoclast, a synovial cell, a meniscal cell, and / or a mesenchymal stem cell.

[0026] Feature 8: The method of feature 6, wherein the cell comprises a low NF-Kb pathway activity in response to SDF-1 providing a suppressed catabolic activity, which is operative to affect cell-mediated anabolism in an inflammatory microenvironment.

[0027] Feature 9: The method of feature 6, wherein the cell is included in a pharmaceutically acceptable formulation.

[0028] Feature 10: The method of feature 6, wherein the cell increases a tissue regeneration in the subject compared to an unmodified cell counterpart.

[0029] Feature 11: The method of feature 6, wherein the pharmaceutical formulation is in the form of a suspension of the CXCR4 OE cells.

[0030] Feature 12: The method of feature 6, wherein the tissue comprises a connective tissue, a meniscus and / or a fibrocartilaginous tissue and / or wherein the cell includes a cartilage- derived progenitor cell line (CPC), a tenocyte, a chondrocyte, an adipocyte, an osteoblast, an osteocyte, an osteoclast, a synovial cell, a meniscal cell, and / or a mesenchymal stem cell.

[0031] Feature 13: The method of feature 6, wherein the cell comprises a cartilage-derived progenitor cell line (CPC), a tenocyte, a chondrocyte, an adipocyte, an osteoblast, an osteocyte, an osteoclast, a synovial cell, a meniscal cell, and / or a mesenchymal stem cell.

[0032] Feature 14: The method of feature 6, wherein the administering is via an intra- articular injection at or near an area of injury, and / or wherein the administering includes a superficial injection into tendon and / or ligament.

[0033] Feature 15: A composition for accelerating musculoskeletal connective tissue repair, comprising a cell according to feature 1.

[0034] Feature 16: The composition of feature 15, wherein the cell comprises any cell that comes from connective tissue, such as (but not limited to) a cartilage-derived progenitor cell lineAttorney Docket No.: 405002-554001WO (CPC), a tenocyte, a chondrocyte, an adipocyte, an osteoblast, an osteocyte, an osteoclast, a synovial cell, a meniscal cell, and / or a mesenchymal stem cell.

[0035] Feature 17: The composition of feature 15, wherein the cell comprises a low NF-Kb pathway activity in response to SDF-1 providing a suppressed catabolic activity, which is operative to affect cell-mediated anabolism in an inflammatory microenvironment.

[0036] Feature 18: The composition of feature 15, wherein the cell is included in a pharmaceutically acceptable formulation.

[0037] Feature 19: The composition of feature 15, wherein the cell increases a tissue regeneration in the subject compared to an unmodified cell counterpart.

[0038] Feature 20: The composition of feature 15, further comprising a pharmaceutically acceptable carrier.

[0039] Feature 21: The composition of feature 15, wherein the composition is formulated for parenteral administration.

[0040] Feature 22: A kit for accelerating a tissue repair in a subject in need thereof or in a subject suspected of having a tissue injury, the kit comprising: (1) a pharmaceutical formulation operative for accelerating a tissue repair, the pharmaceutical formulation including: a cell that is CXCR4-OE; and a solvent, powder, gas, or material that is capable of suspending the cell; and (2) a delivery device or configuration capable of delivering the suspended cell to a subject in need thereof.

[0041] Feature 23: The kit of feature 22, wherein the delivery device comprises a micro- needle or an injection needle operative to provide an intra-articular injection; and / or wherein the configuration comprises an injection without a needle; and / or wherein the administering includes a superficial injection into tendon and / or ligament or into an end of a tendon and / or ligament.

[0042] Feature 24: The cell of feature 1, the method of feature 6, or the composition of feature 15, wherein the cell is a CPC cell; and wherein a risk for a development of post-traumatic osteoarthritis (PTOA) is lowered by the cell, the method, and / or the kit compared to an administration of an unmodified CPC cell under the same conditions.

[0043] Feature 25: The cell of feature 1, the method of feature 6, or the composition of feature 15, wherein the cell, the method, or the composition is included in application including a use of one or more CXCR4 OE tenocytes for a tendinopathy.

[0044] Feature 26: The cell of feature 1, the method of feature 6, or the composition of feature 15, wherein SEQ ID NO: 1 is utilized for initialization in producing the CXCR4-OE and / or wherein an expression comprises SEQ ID NO: 2.Attorney Docket No.: 405002-554001WO

[0045] Feature 27: The cell of feature 1, the method of feature 6, or the composition of feature 15, wherein SEQ ID NO: 1 comprises: gtacaaaaaa gcaggctcca ccatggaggg gatcagtata tacacttcag ataactacac cgaggaaatg ggctcagggg actatgactc catgaaggaa ccctgtttcc gtgaagaaaa tgctaatttc aataaaatct tcctgcccac catctactcc atcatcttct taactggcat tgtgggcaat ggattggtca tcctggtcat gggttaccag aagaaactga gaagcatgac ggacaagtac aggctgcacc tgtcagtggc cgacctcctc tttgtcatca cgcttccctt ctgggcagtt gatgccgtgg caaactggta ctttgggaac ttcctatgca aggcagtcca tgtcatctac acagtcaacc tctacagcag tgtcctcatc ctggccttca tcagtctgga ccgctacctg gccatcgtcc acgccaccaa cagtcagagg ccaaggaagc tgttggctga aaaggtggtc tatgttggcg tctggatccc tgccctcctg ctgactattc ccgacttcat ctttgccaac gtcagtgagg cagatgacag atatatctgt gaccgcttct accccaatga cttgtgggtg gttgtgttcc agtttcagca catcatggtt ggccttatcc tgcctggtat tgtcatcctg tcctgctatt gcattatcat ctccaagctg tcacactcca agggccacca gaagcgcaag gccctcaaga ccacagtcat cctcatcctg gctttcttcg cctgttggct gccttactac attgggatca gcatcgactc cttcatcctc ctggaaatca tcaagcaagg gtgtgagttt gagaacactg tgcacaagtg gatttccatc accgaggccc tagctttctt ccactgttgt ctgaacccca tcctctatgc tttccttgga gccaaattta aaacctctgc ccagcacgca ctcacctctg tgagcagagg gtccagcctc aagatcctct ccaaaggaaa gcgaggtgga cattcatctg tttccactga gtctgagtct tcaagttttc actccagctg aatccaccca gctttcttgt ac, or (GenBank: EU831811.1, 1102 bp, DNA linear SYN, artificial sequence).

[0046] Feature 28: The cell of feature 1, the method of feature 6, or the composition of feature 15, wherein SEQ ID NO: 2 comprises: megisiytsd nyteemgsgd ydsmkepcfr eenanfnkif lptiysiifl tgivgnglvi lvmgyqkklr smtdkyrlhl svadllfvit lpfwavdava nwyfgnflck avhviytvnl yssvlilafi sldrylaivh atnsqrprkl laekvvyvgv wipallltip dfifanvsea ddryicdrfy pndlwvvvfq fqhimvglil pgivilscyc iiisklshsk ghqkrkalkt tvililaffa cwlpyyigis idsfilleii kqgcefentv hkwisiteal affhcclnpi lyaflgakfk tsaqhaltsv srgsslkils kgkrgghssv stesesssfh ss, or (amino acid sequence, GenBank: ACE87329.1, artificial sequence, 352 AA).

[0047] According to some aspects, a kit herein also includes instructions for use. In some examples, a delivery device comprises a micro-needle or an injection needle operative to provide an intra-articular injection, or wherein the configuration comprises an injection without a needle.Attorney Docket No.: 405002-554001WO

[0048] In the cells disclosed herein, the methods, and the kits, a risk for a development of post-traumatic osteoarthritis (PTOA) is lowered by the cell, the method, and / or the kit compared to an administration of an unmodified CPC cell under the same conditions. As such, it is important to note that hypertrophic differentiation is not only the terminal process of endochondral ossification in the growth plate but is also an important pathological change in osteoarthritic cartilage.

[0049] The technology disclosed herein can be applied to any location in a subject, for example, the knee, to wrist, acromioclavicular, sternoclavicular, and temporomandibular joints, and it is contemplated that healing in other areas (such as central nervous system) can be improved as well.

[0050] Thus, those skilled in the art will appreciate that the conception, upon which this disclosure is based, may readily be utilized as a basis for the designing of other structures, methods, and systems for carrying out the various purposes of the present design. It is important, therefore, that the claims be regarded as including such equivalent constructions in so far as they do not depart from the spirit and scope of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] A selection of the novel features of this patent application are in the claims. However, the application itself, as well as a preferred mode of use, and further objectives and advantages thereof, can be introduced by reference to the following detailed description when read in conjunction with the drawings.

[0052] FIG.1 (Prior art) is a plot illustrating how intra-articular injection of CPC stimulates fibrocartilage tear healing. Open areas remaining within the meniscal tear were quantified by image analysis. Data points above the dotted line indicate menisci broken in two at the harvest time. A 0- week control group was used to determine the open space of the meniscal tear before healing. N ≥ 8 per group. ***, P≤0.005.

[0053] FIGs. 2A-2F provide data showing how overexpression of CXCR4 promotes meniscus tear filling and prevents cartilage degeneration. Histology of the meniscus (FIG.2A) and the tibial plateau (FIG.2B) of Saline controls, Unmodified-CPCs treated, and CXCR4-OE CPCs treated animals is shown. Grey arrows (FIG.2B) represent areas of cartilage damage. The percentage of animals with healed vs torn menisci (FIG.2C). Modified OARSI scoring of tibial plateaus (D). N = 6 animals per group. * = P ≤ 0.05. FIG.2D shows OARSI histopathological scoring results. FIG.2E shows how medial side of the femoral condyles were also sectioned, stained, and histologically scored (FIG.2F). * p ≤ 0.05 indicates statistical significances between groups. N = 6 animals per group.Attorney Docket No.: 405002-554001WO

[0054] FIG.3 (Prior Art) is an illustration of an example method used to isolate and immortalize healthy (non-arthritic) human CPC (cartilage-derived progenitor cell) cell lines.

[0055] FIG.4A shows an exemplary creation of a CXCR4-OE-hCPC line. In FIG.4A, the hCPC cell line, which is currently disclosed under the existing patent publication WO2017151646A2, which is incorporated herein by reference in its entirety, was modified further to overexpress the human CXCR4 gene. In FIG.4B and FIG.4C the cells were also tagged with GFP for visualization and tracking. Control (unmodified) cells were generated for comparison, which contained a simple RFP gene tag in place of the human CXCR4 transgene gene construct. (FIG.4D) Western blot was used to confirm successful overexpression of CXCR4, compared to wild-type control cells as well as the RFP expressing control cells. (FIG.4E, “Cell survival assay”) An MTT assay was used to confirm that CXCR4 overexpression did not reduce cell viability. The “ns” (see FIG.4E) refers to non-statistically significant differences between the indicated groups.

[0056] FIG.5 provides an exemplary vector map for implementation in the presently disclosed innovation; this is the expression construct that was delivered to CPCs by lentiviral delivery to generate CXCR4 OE CPCs. See Examples below.

[0057] FIG.6A shows a graphical representation of cell migration assay workflow using damaged / diced rabbit meniscal tissue (10 mg) as a migratory target for cells. Cells placed in the top chamber that are attracted to the tissue target below migrate to the other side of the 8.0 µm pore mesh, where they are harvested and quantified. FIG.6B shows quantitative results of the migration assay analyzing the trafficking of unmodified hCPC and CXCR4-OE-hCPC in the presence and absence of damaged rabbit meniscal tissue. FIG.6C shows a graphical representation of cell migration assay workflow using a collagen 1 scaffold pre-soaked in rSDF-1 (100ng / mL) as the migratory target. FIG.6D shows quantitative results of the migration assay in the presence and absence of SDF-1-soaked collagen scaffold for CXCR4-OE-hCPCs and unmodified hCPCs. FIG.6E shows quantification of soluble SDF-1 released into the supernatants from rabbit menisci (10 mg) and collagen scaffold pre-soaked in rSDF-1. Statistically significant differences are signified as follows: *, p ≤ 0.05; ***, p ≤ 0.001; and ****, p ≤ 0.0001.

[0058] FIG.7 shows examples of data supporting how NF-kB and Erk activity is inhibited in response to SDF-1 stimulation in CPCs. Immunoblot analysis reveals that recombinant SDF-1 treatment reduces inhibitory kinase beta (IkB) levels in BM-MSCs (Left panel) indicating the NF- kB pathway activation. SDF-1 treatment increases IkB in hCPCs (Middle panel) and CXCR4-OE- hCPCs (Right panel). SDF-1 treatment also inhibits Erk pathways in both hCPCs and CXCR4-OE- hCPCs. Opposite results (activation of these pathways) are observed in BM-MSCs (Left panel).Attorney Docket No.: 405002-554001WO MAPK pathway was not significantly modulated upon SDF-1 treatment in BM-MSCs (Left panel), hCPCs (Middle panel) and CXCR4-hCPCs-OE (Right panel).

[0059] FIG.8 shows an example of how an investigation of the therapeutic efficacy of CXCR4-OE-hCPCs was done in vivo, analyzing the effect of these cells in a rabbit meniscus injury model.

[0060] FIG.9A shows an example of creation of immortalized human tenocytes (hTNCL). FIG.9B shows a diagram of creation of hTNCL-CXCR4. FIG.9C shows representative fluorescent microscopy images of control hTNCL (bearing dual reporter) and hTNCL-CXCR4. Images were taken at 10X magnification. FIG.9D shows MTT cell proliferation assay (n=4) n.s. indicates not significant.

[0061] FIG.10A shows a schematic representation of 3D cell migration assay. FIG.10B shows an MTT assay to quantify the migrated cells from the outer side of the 8-micron cell culture insert. Wells without the scaffold were used as controls. *** = p value < 0.001.

[0062] FIG. 11 shows an illustration diagram of CXCR4 overexpression and hCPC function. FIG.12 shows an example method flowchart.

[0063] While the embodiments and method of the present application is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the description herein of specific embodiments is not intended to limit the application to the particular embodiment disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the process of the present application as defined by the appended claims. It is important to note that any example, discussion, disclosure, feature, aspect, embodiment or wording herein can be inter-combined with any other and should not be construed as any limiting wording (or description) in the spirit of improving human lives. DETAILED DESCRIPTION

[0064] Illustrative embodiments of the preferred embodiment are described below. In the interest of clarity, not all features of an actual implementation are described in this specification. It will of course be appreciated that in the development of any such actual embodiment, numerous implementation-specific decisions must be made to achieve the developer’s specific goals, such as compliance with system-related and business-related constraints, which will vary from one implementation to another. Moreover, it will be appreciated that such a development effort mightAttorney Docket No.: 405002-554001WO be complex and time-consuming but would nevertheless be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure. DEFINITIONS

[0065] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0066] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.

[0067] As used herein, the term "approximately" or "about" in reference to a value or parameter are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). As used herein, reference to "approximately" or "about" a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X" includes description of "X".

[0068] As used herein, the term “or” means “and / or.” The term "and / or" as used in a phrase such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0069] As used herein, the term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation.

[0070] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.Attorney Docket No.: 405002-554001WO

[0071] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0072] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two-standard deviation (2SD) or greater difference.

[0073] As used herein, the term "subject" refers to a mammal, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. Subjects can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), but are not so limited. Subjects include human subjects. The human subject may be a pediatric, adult, or a geriatric subject. The human subject may be of either sex.

[0074] As used herein, the terms "effective amount" and “therapeutically effective amount” include an amount sufficient to prevent or ameliorate a manifestation of disease or medical condition, such as peripheral nerve injuries, muscle injuries, ligament injuries, and tendon injuries. It will be appreciated that there will be many ways known in the art to determine the effective amount for a given application. For example, the pharmacological methods for dosage determination may be used in the therapeutic context. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds.

[0075] As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” when used in reference to a disease, disorder or medical condition, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally “effective” if one or more symptoms or clinical parameters are improved. Alternatively, treatment is “effective” if the progression of a condition is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or clinical parameters, but also a cessation or at least slowing down of progression or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or moreAttorney Docket No.: 405002-554001WO symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of peripheral nerve injuries.

[0076] As used herein, the term "long-term" administration means that the therapeutic agent or drug is administered for a period of at least 12 weeks. This includes that the therapeutic agent or drug is administered such that it is effective over, or for, a period of at least 12 weeks and does not necessarily imply that the administration itself takes place for 12 weeks, e.g., if sustained release compositions or long-acting therapeutic agent or drug is used. Thus, the subject is treated for a period of at least 12 weeks. In many cases, long-term administration is for at least 4, 5, 6, 7, 8, 9 months or more, or for at least 1, 2, 3, 5, 7 or 10 years, or more.

[0077] The administration of the compositions contemplated herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. In a preferred embodiment, compositions are administered parenterally. The phrases “parenteral administration” and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intra-lymphatic, intra-lymph node, intravenous, intraportal vein, intrahepatic arterial, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, intranasal, intratracheal, intrathecal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0078] The terms: “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.Attorney Docket No.: 405002-554001WO

[0079] The terms: “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10- 100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.

[0080] As used herein, articular cartilage-derived progenitor cell lines (CPCs) refer to cell lines derived from a subject and utilized for an accelerated treatment. In general, CPCs can be characterized by one or more of such things as stem cell markers, immortalization, multi-lineage ability, and self-renewal potential. These may be found in different cartilage tissues, such as auricular cartilage, articular cartilage, and nasal cartilage.

[0081] As used herein, stromal cell-derived factor 1 (SDF-1) refers to a protein that can occur in various forms by, for example, alternate splicing of the same gene from which is arises. In some embodiments, SDF-1 can also be known as C-X-C motif chemokine 12 (CXCL12) protein, which is a chemokine protein that in humans is encoded by the CXCL4 gene. The gene is known to produce 7 isoforms through alternative splicing. SDF-1 pathway signaling is necessary for stimulating the directional migration of CPCs to stimulate meniscal fibrocartilage repair, and these stem / progenitor cells are thought to respond to injury and migrate into cartilage defect zones.

[0082] It is contemplated that the technology herein can be utilized in combination with any other technology such as combination therapies, with visualization tools, with nanoparticles, with antibodies (e.g., for other targeting methods), with magnetics or electromagnetic radiation), with other genetic modifications / cells; and as such the embodiments discussed herein do not limit the future scope contemplated for the technology. PHARMACEUTICAL COMPOSITIONS

[0083] The compositions and methods of the present invention may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueousAttorney Docket No.: 405002-554001WO solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0084] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-micro emulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0085] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0086] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars,Attorney Docket No.: 405002-554001WO such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0087] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Patent Nos.6,110,973; 5,763,493; 5,731,000; 5,541,231; 5,427,798; 5,358,970; and 4,172,896, as well as in patents cited therein.

[0088] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

[0089] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared byAttorney Docket No.: 405002-554001WO uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0090] Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

[0091] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragées, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0092] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.Attorney Docket No.: 405002-554001WO

[0093] The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragées, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropyl methyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

[0094] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, micro-emulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0095] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0096] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0097] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compoundAttorney Docket No.: 405002-554001WO may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

[0098] The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0099] Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0100] Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

[0101] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0102] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol,Attorney Docket No.: 405002-554001WO and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0103] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0104] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0105] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

[0106] For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0107] Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.Attorney Docket No.: 405002-554001WO

[0108] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0109] The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0110] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art.D

[0111] In general, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0112] If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the active compound may be administered two or three times daily. In other embodiments, the active compound will be administered once daily.Attorney Docket No.: 405002-554001WO

[0113] The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines bovine, porcine, sheep, feline, and canine; poultry; and pets in general.

[0114] In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent.

[0115] The present disclosure includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, 1-hydroxy-2- naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4- acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid salts.

[0116] The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.Attorney Docket No.: 405002-554001WO

[0117] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0118] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha- tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. EXEMPLARY SEQUENCES

[0119] The sequences for associated peptides, proteins, oligonucleotides, and various RNA / DNA, viruses, vectors, cells, or combinations thereof are incorporated by reference herein as these are known, for example, a CXCR4 gene is shown at ncbi.nlm.nih.gov / gene / 7852 (for humans). The technology disclosed herein includes the sequences required to practice the Invention.

[0120] In another example, the mRNA was derived from the Synthetic construct Homo sapiens clone HAIB:100066840; DKFZo008F0721 chemokine (C-X-C motif) receptor 4 protein (CXCR4) gene, (encodes complete protein), and is shown below as SEQ ID NO: 1 (GenBank: EU831811.1, 1102 bp, DNA linear SYN, artificial sequence), as shown at: https: / / www.ncbi.nlm.nih.gov / nuccore / EU831811. 0001 gtacaaaaaa gcaggctcca ccatggaggg gatcagtata tacacttcag ataactacac 0061 cgaggaaatg ggctcagggg actatgactc catgaaggaa ccctgtttcc gtgaagaaaa 0121 tgctaatttc aataaaatct tcctgcccac catctactcc atcatcttct taactggcat 0181 tgtgggcaat ggattggtca tcctggtcat gggttaccag aagaaactga gaagcatgac 0241 ggacaagtac aggctgcacc tgtcagtggc cgacctcctc tttgtcatca cgcttccctt 0301 ctgggcagtt gatgccgtgg caaactggta ctttgggaac ttcctatgca aggcagtcca 0361 tgtcatctac acagtcaacc tctacagcag tgtcctcatc ctggccttca tcagtctgga 0421 ccgctacctg gccatcgtcc acgccaccaa cagtcagagg ccaaggaagc tgttggctga 0481 aaaggtggtc tatgttggcg tctggatccc tgccctcctg ctgactattc ccgacttcat 0541 ctttgccaac gtcagtgagg cagatgacag atatatctgt gaccgcttct accccaatga 0601 cttgtgggtg gttgtgttcc agtttcagca catcatggtt ggccttatcc tgcctggtat 0661 tgtcatcctg tcctgctatt gcattatcat ctccaagctg tcacactcca agggccacca 0721 gaagcgcaag gccctcaaga ccacagtcat cctcatcctg gctttcttcg cctgttggct 0781 gccttactac attgggatca gcatcgactc cttcatcctc ctggaaatca tcaagcaaggAttorney Docket No.: 405002-554001WO 0841 gtgtgagttt gagaacactg tgcacaagtg gatttccatc accgaggccc tagctttctt 0901 ccactgttgt ctgaacccca tcctctatgc tttccttgga gccaaattta aaacctctgc 0961 ccagcacgca ctcacctctg tgagcagagg gtccagcctc aagatcctct ccaaaggaaa 1021 gcgaggtgga cattcatctg tttccactga gtctgagtct tcaagttttc actccagctg 1081 aatccaccca gctttcttgt ac

[0121] Regarding an example of the translated protein, the chemokine (C-X-C motif) receptor 4 protein [synthetic construct] is shown as SEQ ID NO: 2 (amino acid sequence, GenBank: ACE87329.1, artificial sequence, 352 AA) below, and is also shown at: https: / / www.ncbi.nlm.nih.gov / protein / 190691109. 001 megisiytsd nyteemgsgd ydsmkepcfr eenanfnkif lptiysiifl tgivgnglvi 061 lvmgyqkklr smtdkyrlhl svadllfvit lpfwavdava nwyfgnflck avhviytvnl 121 yssvlilafi sldrylaivh atnsqrprkl laekvvyvgv wipallltip dfifanvsea 181 ddryicdrfy pndlwvvvfq fqhimvglil pgivilscyc iiisklshsk ghqkrkalkt 241 tvililaffa cwlpyyigis idsfilleii kqgcefentv hkwisiteal affhcclnpi 301 lyaflgakfk tsaqhaltsv srgsslkils kgkrgghssv stesesssfh ss MOTILE INJECTABLE CELLS THAT ACCELERATE MUSCULOSKELETAL CONNECTIVE TISSUE REPAIRS

[0122] In a broad description of the Invention herein, a genetically engineered injectable chondroprogenitor cell line is disclosed that accelerates healing of proteoglycan containing connective tissues (i.e., cartilage, fibrocartilage). This cell line has taken years to develop for the effectiveness that is demonstrated below.

[0123] A critical problem addressed by the Invention is that musculoskeletal healing following a traumatic injury involves the migration and localization of satellite progenitor cells residing in the tissue. These cells differentiate and coordinate the healing process making progenitor cell trafficking a limiting factor for injury recovery. Our invention has demonstrated efficacy for accelerating cartilage connective tissue healing.

[0124] How it works: According to some aspects, the invention is a Stromal Cell Derived Factor Inducible Chondroprogenitor Cell Line (SDF-iCPCL) – an immortalized (stable) mesenchymal chondroprogenitor cell line that is genetically modified to overexpress the human CXCR4 gene. CXCR4 is the receptor to the chemokine stromal cell derived factor (SDF-1). The cellular expression of CXCR4 in these cells accomplish three pivotal goals: (1) increases the migration of SDF-iCPCL towards cartilage tissue (including fibrocartilage) injury sites; (2) diminishes matrix degradative catabolic pathway activities; (3) increases cellular anabolism of extracellular matrix proteins that are essential for rebuilding cartilage tissues. Upon reaching theAttorney Docket No.: 405002-554001WO injury site, SDF-iCPCL will engraft into the tissue and accelerate healing of the injured cartilage tissues.

[0125] Advantages over Existing Technologies: Currently, the clinical standards for cartilage tissue repair includes 1) Suturing the injury site; 2) replacing damaged tissue with allograft; 3) bone marrow stimulation to promote mesenchymal stromal cell migration into the joint space; 4) implantation of autologous chondrocytes to fill tissue lesions. All of these are highly invasive procedures that require surgery. Our invention has an advantage over these procedures because SDF-iCPCL can be injected into the joint space (intraarticularly), at which point they will chemotactically migrate to the injury site and improve healing. This is a minimally invasive injectable cellular biologic that can revolutionize how joint injuries are treated. Additionally, because SDF-iCPCL diminishes matrix degradative catabolic pathway activities and increases cellular anabolism of extracellular matrix proteins that are essential for rebuilding cartilage tissues, it makes them unique in their response to SDF-1 ligand. Prior art has shown that SDF-1 instigates cellular catabolism in cells (including other mesenchymal progenitor cell subsets), while SDF- iCPCL acts in the opposite fashion.

[0126] After considering the embodiments above, it is important to note that this invention can be used to accelerate cartilage and fibrocartilage tissue healing, and this invention can be leveraged to stimulate cartilage healing in human patients and in animals. Meniscus injuries that fail to heal can instigate catabolic changes in the knee microenvironment, posing a high-risk for the development of post-traumatic osteoarthritis (PTOA). We have established human articular cartilage-derived progenitor cell lines (CPCs) as a potential therapeutic tool in our continuous efforts to develop novel cell-based approaches for accelerating meniscus healing. Further, characterization of these cell lines revealed that they have several pro anabolic and anti-catabolic properties in comparison to marrow-derived stromal cells (BM- MSCs)E,F,G.

[0127] Veritably, our most recent in vivo data suggest that treating meniscus tears with a human CPC cell line promotes meniscal tear healing compared to either the BM-MSC-treated group or untreated control group in athymic rats (FIG.1)I. In FIG.1, (see Desai, et al.I, at page 7, Figure 3E) intra-articular injection of CPC stimulates fibrocartilage tear healing with lower defect areas (µm2) compared to BM-MSCs. Additionally, we had demonstrated that Stromal Cell-Derived Factor-1 (SDF-1) pathway signaling is necessary for stimulating the directional migration of CPCs to stimulate meniscal fibrocartilage repairE,F. SDF-1 is highly expressed by injured meniscus tissueH, making it crucially important in the context of how cells used in biologic therapies react to the presence of this chemokine. In this study, we demonstrate that CPCs exhibit reduced NF-kBAttorney Docket No.: 405002-554001WO catabolic pathway signaling in response to SDF-1. Hence, we hypothesized that constitutively increasing the expression of CXCR4 (an SDF-1 receptor) in CPCs may further improve their efficacy in meniscus injury repair. Our findings below (e.g., Examples) show that administering CXCR4 overexpressing CPCs via intraarticular injection significantly improved meniscus tear healing and protects against cartilage erosion in rabbits. It is critical to note that exemplary improvements of this technology (CXCR4 OE CPCs) over the basic technology (unmodified CPCs) disclosed in Desai, et al., 2022 include:

[0128] ^ CXCR4 OE CPCs are more chemotactic (superior at migrating towards injured meniscus tissue).

[0129] ^ CXCR4 OE CPC injection into immunocompetent animals (animals with an uncompromised intact immune system) achieve superior meniscal healing, and protects against cartilage erosion. The unmodified CPC line (which was the subject of Desai, et al., 2022) was less effective. For example, see FIG.2A, FIG.2B, FIG.2C, and FIG.2D.

[0130] ^ CXCR4 OE CPCs are tagged with fluorescent green protein (GFP) for easy in vitro and in vivo cell tracking. For example, see FIG.4B and FIG.4C (GFP).

[0131] Briefly, in the Scientific Examples presented below, healthy CPC cell lines are infected with lentivirus bearing CXCR4 followed by Puromycin selection and expansion. These cell lines are then utilized in approved animal studies to demonstrate the technology. The resulting data show that the SDF-1 / CXCR4 pathway regulation was investigated in CPCs. To investigate the mechanistic details, we examined the expression of downstream targets of these canonical pathways in response to SDF-1. We analyzed the NF-kB pathway by measuring the protein expression of IkB-α and NF-kB p65. The Erk and MAPK pathway were analyzed by measuring Erk and p38 along with their phosphorylation states (pErk) and (p-p38). In some embodiments, our results indicate that SDF-1 treatment in CPCs leads to significant inhibition of canonical NF-κB (compare FIG.7) Erk and MAPK, which are upregulated in response to SDF-1 in other cell types. Our results from the rabbit study demonstrate that CXCR4-OE CPCs significantly improve meniscus tear healing as compared to the unmodified-CPC or saline controls (FIG.2A) (see the circled region of interest). Additionally, CXCR4-OE CPCs promote restoration of proteoglycan content as can be seen by Saf-O (red) staining (FIG.2A, also FIG.2B). We carefully examined all the menisci (18 total; 6 per experimental group) for healing (FIG.2C), which indicates that 83% animals showed fully healed menisci in CXCR4 OE CPCs treated group, which was significantly high as compared to Unmodified-CPC treated (50%) and saline control treated animals (33%). Additionally, CXCR4-OE CPCs significantly prevents articular cartilage degradation in tibial plateau, as compared to the saline controls (FIG.2B, FIG.2D).Attorney Docket No.: 405002-554001WO

[0132] Discussion: Our results demonstrate that intra-articular injection of CPCs following meniscus tearing stimulates fibrocartilage restoration and healing in athymic ratsI. Previously, we have demonstrated that SDF1 / CXCR4 signaling axis is crucial for migration of CPCs on the injury site in vivoF. To investigate the mechanistic details, we examined the SDF-1 / CXCR4 downstream signaling pathway by analyzing different catabolic branches of the pathway including NF-kB, Erk and MAPK. Our results revealed that these catabolic signaling is inhibited in CPCs in response to SDF-1 treatment. These findings are consistent with our previous observation that matrix metalloproteinase 13 (MMP13), a downstream target of NF-κB, is maintained at a lower expression level in CPCs than in BM-MSCsI. Additionally, CPCs that overexpress CXCR4 chemokine significantly promotes meniscus tear healing, restores proteoglycan content, and prevents cartilage degeneration in rabbits. Collectively these findings support our hypothesis that CXCR4 plays a vital role in CPCs mediated meniscus and cartilage repair through inhibiting downstream, catabolic, and inflammatory signaling.

[0133] Significance / Clinical Relevance: This study demonstrates, with enabling data, that stable human CPCs made to overexpress CXCR4 can be used as an injectable cell therapy to stimulate meniscus healing and prevent PTOA in rabbits.

[0134] The technology can be commercially distributed. In some embodiments, a kit for accelerating a musculoskeletal connective tissue repair in a subject in need thereof or in a subject suspected of having a musculoskeletal connective tissue injury is disclosed herein, the kit comprising: (1) a pharmaceutical formulation operative for accelerating a musculoskeletal connective tissue repair, the pharmaceutical formulation including: a CPC cell that is CXCR4-OE; and a solvent, powder, gas, or material that is capable of suspending the cell; and (2) a delivery device or configuration capable of delivering the suspended cell to a subject in need thereof. According to some aspects, the kit also includes instructions for use. In some examples, the delivery device comprises a micro-needle or an injection needle operative to provide an intra- articular injection, or wherein the configuration comprises an injection without a needle.

[0135] According to some aspects, the technology herein provides a cell that accelerates a musculoskeletal connective tissue repair, the cell including a genetic modification such that the cell is a CXCR4 overexpressing (CXCR4-OE) cell. In some embodiments, the cell is a motile cell that can be administered to a subject in need thereof; and the cell accelerates musculoskeletal connective tissue repair compared to the same cell line that is not carrying the genetic modification. In eamples discussed herein, the cell comprises a cartilage-derived progenitor cell line (CPC). In some embodiments, a pharmaceutical formulation comprising the cell discussed above is provided.Attorney Docket No.: 405002-554001WO

[0136] In some embodiments, the cell discussed above has a low NF-Kb pathway activity in response to SDF-1 (i.e., this means it has suppressed catabolic activity, which is important for cell-mediated anabolism especially in an inflammatory microenvironment, like the injured knee).

[0137] According to some aspects, a method of treating a subject diagnosed with a musculoskeletal connective tissue injury or a subject suspected of having a musculoskeletal connective tissue injury is disclosed herein, the method comprising the steps of: (1) obtaining a pharmaceutical formulation comprising a plurality of cells comprising a genetic modification such that the cells are CXCR4 overexpressing (CXCR4-OE); and (2) administering a therapeutically effective amount of the cells via administering the pharmaceutical formulation. In this example of the method, a healing or repair of the musculoskeletal connective tissue injury is accelerated compared to a treating using the same formulation without the CXCR4-OE.

[0138] In the methods and the pharmaceutical formulations, in some embodiments, the pharmaceutical formulation is in the form of a suspension of the cells.

[0139] According to some aspects, the connective tissue comprises any connective tissue in need of accelerated healing. In some embodiments, the administering to the subject is via any path of administration known in the art.

[0140] In the methods and formulations, according to some aspects, the cell comprises a cartilage-derived progenitor cell line (CPC).

[0141] In the cells disclosed herein, the methods, and the kits, it is contemplated that a risk for a development of post-traumatic complications is lowered, thereby improving long-term outcomes for patients.

[0142] In an overview of data supporting the embodiments in the Summary and Detailed Description, CPCs were genetically modified to overexpress gene CXCR4 and the resulting proteins were analyzed. New Zealand White Rabbits were injured then treated with the modified CPCs. Compared to controls, the treatments worked highly effectively (see Examples below). INTRAARTICULAR INJECTION OF CXCR4 OVEREXPRESSING HUMAN CPCS IMPROVES MENISCUS HEALING AND PROTECTS AGAINST PTOA IN IMMUNOCOMPETENT RABBITS

[0143] In a description of further investigations, a background setting is described as follows: Meniscus injuries that fail to heal instigate catabolic changes in the knee microenvironment, posing a high risk for developing post-traumatic osteoarthritis (PTOA). Previous research suggests that human cartilage-derived progenitor cells (hCPCs) can stimulate meniscus tear repair in a manner that depends on Stromal Cell-Derived Factor-1 (SDF-1) pathway activity.

[0144] According to some aspects, the technology provides a novel CXCR4 overexpressing cell for enhanced musculoskeletal connective tissue repair. In the field ofAttorney Docket No.: 405002-554001WO regenerative medicine, various approaches have been explored to enhance the repair of musculoskeletal connective tissues, such as tendons, ligaments, and cartilage. Traditional methods often involve surgical interventions, physical therapy, and the use of growth factors or scaffolds to promote tissue regeneration. While these methods can be effective, they often require long recovery times and may not fully restore the function of the damaged tissue. Additionally, the use of growth factors can sometimes lead to undesirable side effects, such as inflammation or abnormal tissue growth.

[0145] Cell-based therapies have emerged as a promising alternative for musculoskeletal tissue repair. These therapies typically involve the use of stem cells or progenitor cells that can differentiate into the desired cell type and contribute to tissue regeneration. Mesenchymal stem cells (MSCs), for example, have been widely studied for their potential to repair connective tissues due to their ability to differentiate into osteoblasts, chondrocytes, and tenocytes. However, the efficacy of MSCs can be limited by their homing ability to the site of injury and their survival in the harsh microenvironment of damaged tissues.

[0146] Genetic modification of cells to enhance their regenerative capabilities has also been investigated. Techniques such as overexpressing certain genes or silencing others have been employed to improve cell survival, proliferation, and differentiation. For instance, the overexpression of growth factor receptors or anti-apoptotic genes has been shown to enhance the therapeutic potential of cells used in tissue repair. Despite these advancements, challenges remain in achieving efficient and targeted delivery of genetically modified cells to the injury site, as well as ensuring their sustained activity and integration into the host tissue. This disclosure combines features, details, aspects, embodiments, and examples to provide new solutions.

[0147] The invention relates to a genetically modified cell designed to accelerate tissue repair. The tissue repair can be, for example, musculoskeletal connective tissue repair. The cell is characterized by overexpression of the CXCR4 receptor, rendering it a CXCR4 overexpressing (CXCR4 OE) cell. This modification enhances the cell's motility, allowing it to be effectively administered to subjects requiring tissue repair. The CXCR4 OE cell demonstrates a significant improvement in accelerating the repair of musculoskeletal connective tissues compared to its non- genetically modified counterparts. This advancement offers potential therapeutic benefits in treating injuries or conditions affecting musculoskeletal connective tissues, providing a novel approach to enhancing tissue regeneration and recovery.

[0148] Hypothesis: Overexpressing the SDF-1 receptor CXCR4 in CPCs will increase cell trafficking and further improve their repair efficacy of meniscal injuries. Methods: HCPCs were genetically modified to overexpress CXCR4 (CXCR4-OE-hCPCs) using lentivirus. In vitroAttorney Docket No.: 405002-554001WO characterization was performed using cell viability assay, cell migration assay, and immunoblotting. These cells were then used to treat a meniscal injury in rabbits. A medial meniscal tear was surgically created in the right knees of New Zealand White Rabbits followed by two intraarticular injections (5.0×10^6 cells each) of either CXCR4-OE-hCPCs, wild-type hCPCs, or just saline alone. Histological assessment of the menisci and cartilage was performed using Saf O / Fast green stained. Joints were assessed for PTOA changes using modified OARSI scoring.

[0149] Fluorescent imaging and DNA analysis was performed to interrogate tissues for human cells. Results: SDF-1 inhibits NF-kB and ERK pathways in both wild-type and CXCR4-OE- hCPCs. CXCR4 overexpression increased hCPC trafficking towards sources of SDF-1, including injured meniscal fibrocartilage and an SDF-1 presoaked collagen scaffold. Intraarticular injection of CXCR4-OE-hCPCs significantly improved meniscus tear healing as evidenced by the complete absence of tear in 5 out of 6 (83%) animals that received CXCR4-OE-hCPCs as compared to only 3 out of 6 (50%) in control hCPC treated animals, and 2 out of 6 (33%) animals in the saline control group. CXCR4-OE-hCPC-treated animals also showed significantly less erosion in their knee cartilage, compared to control animals. Conclusion: Overall, CXCR4 overexpression inhibited catabolic pathway signaling in hCPCs and increased cell migration. Evidence suggests that intraarticular injection of these cells into the injured knee allows them to home in on sites of fibrocartilage injury and ultimately results in meniscal tear healing and PTOA inhibition in immunocompetent animals. Clinical Relevance: This study demonstrates that cartilage progenitors with elevated CXCR4 expression has the potential to be a potent therapeutic tool for stimulating meniscus tear healing.

[0150] Meniscal tears are common and they can increase the risk of osteoarthritis, especially due to their high rate of reinjury following clinical treatment. In the last decade, cell- based musculoskeletal tissue repair strategies have shown great promise pre-clinically, demonstrating that they have the potential to revolutionize orthopaedic medicine. There exists a need to develop a safe and effective cell-based strategy that can stimulate meniscus healing and minimize the need for reoperation to prevent OA following these traumatic injuries. Some examples of what this study adds to the existing knowledge: In this study, we report on the pre-clinical efficacy of a novel and minimally invasive strategy for stimulating meniscal tear reintegration. Here, we use intra-articular injection of a genetically modified human progenitor cell line, demonstrated to have heightened chemoattraction towards injured fibrocartilage tissues, to promote meniscal tear healing and inhibit PTOA in a rabbit model of meniscal injury. Lastly, we discover that these cells respond uniquely to the pro-inflammatory chemokine SDF-1 in a manner that helps to explain their anabolic effects, despite the inflammatory microenvironment present in the injured knee.Attorney Docket No.: 405002-554001WO

[0151] The menisci are semicircular crescent-shaped fibrocartilaginous pads situated between the femoral condyle and tibial plateau of the knee joint14. Meniscus injuries that are left untreated impart catabolic changes in the knee microenvironment,5which ultimately results in the development of post-traumatic osteoarthritis (PTOA)11,12,32,40. Currently, acute meniscus injury is treated either non-surgically (physiotherapy or NSAIDs), or by suturing, partial / complete meniscectomy, or replacement. Although these approaches improve patients’ quality of life in the short term, they do not eliminate the risk of patients developing accelerated post-traumatic osteoarthritis (PTOA) in the long term;3,7,12hence, there is room for improvement. There exists a need to develop more efficient minimally invasive therapeutic strategies for meniscus injuries and PTOA prevention.

[0152] Stem cell therapeutics have gained much attention from the scientific community in the past two decades for being a promising approach that can revolutionize how we treat musculoskeletal tissue injuries2,17-19. Stem / progenitor cells from different origins, including meniscal fibrocartilage, articular cartilage, and marrow have been investigated for meniscus repair applications4,9,22,30. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are the most commonly investigated cells for this purpose in pre-clinical animal models because of their, bioavailability, extensive proliferation capability and plasticity25,43. However, BM-MSCs have certain limitations. During late-stage chondrogenesis, BM-MSCs exhibit increased gene expression of common cartilage hypertrophy-ossification markers, which pose a significant challenge for cartilage tissue engineering28,37.

[0153] A recent study has demonstrated the therapeutic reparative potential of using healthy human articular cartilage-derived mesenchymal progenitor cells (hCPCs) for meniscus repair in explant organ culture models18,29as well as in an immunocompromised rodent model of meniscus injury8. Molecular and phenotypic characterization of these cells revealed that hCPCs are highly proliferative and chondrogenic with anti-catabolic properties in comparison to bone- marrow-derived mesenchymal stem cells (BM-MSCs)16-18. Additionally, it has been demonstrated that Stromal Cell-Derived Factor-1 (SDF-1) pathway signaling is necessary for stimulating the directional migration of CPCs to stimulate meniscal fibrocartilage repair18,29. SDF-1 is a chemokine that is highly expressed by injured meniscus tissue24,36,39and it is crucial for the targeted migration of cells to sites of injury. The most well-known SDF-1 receptor is CXCR4, which operates through complex downstream signaling that regulates multiple processes including cell survival, cell migration, inflammation, and matrix remodeling6,31.

[0154] Based on this background, we hypothesize that overexpression of CXCR4 may enhance the retention of cells at the injury site, thereby facilitating a more efficient repair processAttorney Docket No.: 405002-554001WO for meniscal injuries. This retention could lead to prolonged local activity of cells, ultimately improving the regenerative outcomes associated with meniscus and cartilage damage. To achieve this aim, we genetically modified hCPCs to stably overexpress CXCR4 (CXCR4-OE-hCPCs). We characterized CXCR4 downstream pathway signaling in these cells and evaluated their cell migration capability. Lastly, we tested their therapeutic efficacy for stimulating meniscus healing in a fully immunocompetent animal model. Our findings revealed that CPCs exhibit reduced NF-kB catabolic pathway signaling in response to SDF-1, which is a unique characteristic that is not exhibited by other cell types, including BM-MSCs. We also observed that CXCR4-OE-hCPCs significantly outperformed wild-type hCPCs in terms of chemotactic migration towards damaged meniscal fibrocartilage as well as their propensity for stimulating meniscus tear healing. Finally, we found that CXCR4-OE-hCPC-treated animals exhibited significantly less erosion of the articular cartilage surface of the femoral condyle as well as the tibial plateau as compared to the control animals.

[0155] See Example 3, In Vitro Assays below for more experimental information. Results— Establishment and Characterization of hCPC-CXCR4-OE: We have established hCPCs cell lines constitutively overexpressing CXCR4 using lentiviral vectors (see FIG.4A) carrying the Green Fluorescent Protein reporter gene downstream to CXCR4 and hence express GFP as an indicator of CXCR4 overexpression. In the non-targeting controls, the CXCR4 gene was replaced with Turbo-RFP and hence carried dual (Red + Green) fluorescence (FIG.4B). We confirmed the overexpression of CXCR4 by the presence of green fluorescence (FIG.4C). The overexpression was further validated by immunoblotting (FIG.4D). We then investigated the effect of lentiviral infection and CXCR4 overexpression on the viability of the cells at passage 7, post-infection, by cell survival assay using MTT reagent. Our results indicate that the viability of unmodified control hCPCs and CXCR4-OE-hCPCs is not affected significantly upon infection. Moreover, overexpression of CXCR4 does not adversely affect the cell viability of hCPCs (FIG.4E).

[0156] In these examples, FIGs. 4A-4E show examples of establishment and characterization of CXCR4-OE-hCPCs. (FIG.4A) Schematic of how CXCR4-OE-hCPCs were generated using lentiviral transduction, and how they were used to conduct characterization and in vivo efficacy experiments. (FIG.4B) Graphical representation of lentiviral backbone carrying GFP downstream to CXCR4. For the unmodified control hCPCs, the CXCR4 gene cassette was replaced with RFP rendering the cells dual fluorescent. (FIG.4C) Representative image of hCPCs overexpressing CXCR4 (green fluorescence) (Right panel) and non-targeting control (red and green dual fluorescence) (Left panel). Images were taken at 20× Magnification using a Nikon inverted phase contrast microscope. (FIG.4D) Immunoblot analysis of CXCR4 protein confirmingAttorney Docket No.: 405002-554001WO its overexpression in CXCR4-OE-hCPCs. β-Actin was used as an internal loading control. (FIG.4E) Cell survival assay performed using MTT reagent shows no changes in cell viability following viral transduction and CXCR4 overexpression. ns = p ≥ 0.5 (not significant).

[0157] SDF-1 promotes migration of hCPCs-CXCR4-OE: To examine the effect of SDF-1 on the cell trafficking of hCPCs and CXCR4-OE-hCPCs, we performed a cell migration assay using 10 mg of damaged rabbit meniscus tissue as a migratory target (FIG.6A). Cells that had migrated and crossed over to the outer side of the 8-micron membrane were collected and quantified 48 hours following their incubation with the damaged meniscus tissue. CXCR4-OE-hCPCs migrated towards the injured tissue at a significantly higher rate in comparison to the unmodified control hCPCs (FIG.6B). To further confirm that the migration was caused by SDF-1, we performed a similar cell migration assay using a collagen scaffold that was pre-soaked in recombinant SDF-1 (rSDF-1) and observed similar results (FIG.6C, FIG.6D). We confirmed the presence of SDF-1 protein that had been released into the supernatants in both experiments by quantifying soluble SDF-1 via ELISA (FIG.6E). Additionally, ELISA results indicated that 10 mg of damaged rabbit meniscal fibrocartilage can produce up to 20 ng / mL of soluble SDF-1 protein.

[0158] In these data, FIGs.6A-6E show overexpression of CXCR4 significantly enhances cell trafficking of hCPCs. (FIG.6A) Graphical representation of cell migration assay workflow using damaged / diced rabbit meniscal tissue (10 mg) as a migratory target for cells. Cells placed in the top chamber that are attracted to the tissue target below migrate to the other side of the 8.0 µm pore mesh, where they are harvested and quantified. (FIG.6B) Quantitative results of the migration assay analyzing the trafficking of unmodified hCPC and CXCR4-OE-hCPC in the presence and absence of damaged rabbit meniscal tissue. (FIG.6C) Graphical representation of cell migration assay workflow using a collagen 1 scaffold pre-soaked in rSDF-1 (100ng / mL) as the migratory target. (FIG.6D) Quantitative results of the migration assay in the presence and absence of SDF- 1-soaked collagen scaffold for CXCR4-OE-hCPCs and unmodified hCPCs. (FIG.6E) Quantification of soluble SDF-1 released into the supernatants from rabbit menisci (10 mg) and collagen scaffold pre-soaked in rSDF-1. Statistically significant differences are signified as follows: *, p ≤ 0.05; ***, p ≤ 0.001; and ****, p ≤ 0.0001.

[0159] CXCR4-OE-hCPCs maintain NF-kB and Erk pathway inhibition in response to SDF- 1 stimulation: Next, we characterized and compared SDF-1 / CXCR4 downstream signaling in CXCR4-OE-hCPCs and unmodified hCPCs, in the presence and absence of rSDF-1 stimulation. BM-MSCs were also included for comparison, since they are one of the most used cell types in pre-clinical cell-based meniscus repair studies that have been performed previously. We specifically checked for activation of NF-κB, Erk, and p38 MAPK in response to SDF-1 in theseAttorney Docket No.: 405002-554001WO cells since they represent different downstream branches of SDF-1 / CXCR4 downstream signaling (FIG.7). We first analyzed the NF-kB pathway by examining the level of inhibitory kinase – beta (IkB), in the absence and dose-dependent presence of rSDF-1. NF-kB pathway activation is well- known to be associated with inflammation and catabolism. In BM-MSCs, rSDF-1 treatment significantly reduced the protein level of IkB, which primarily indicates NF-kB pathway activation. Surprisingly, the opposite trend was observed in both unmodified hCPCs and CXCR4-OE-hCPCs. Indeed, both types of CPCs did not demonstrate a decrease in inhibitory kinase in response to treatment with rSDF-1, indicating that the NF-κB pathway is being inhibited. Additionally, CXCR4- OE-hCPCs maintained higherbasal IkB levels, even in the absence of rSDF-1.

[0160] In unmodified hCPCs and in CXCR4-OE-hCPCs, rSDF-1 treatment also caused a significant reduction in phospho-Erk and total Erk protein levels. However, BM-MSCs exhibited increases in both of these signaling molecules, in response to rSDF-1. p38 MAPK levels were not significantly altered between BM-MSCs and the CPCs, suggesting that this cell survival pathway is not differently regulated across these cells in response to rSDF-1. Taken together, our data demonstrates that in comparison to BM-MSCs, CPCs exhibit differential regulation of SDF- 1 / CXCR4 downstream signaling with respect to NF-kB and Erk pathway activity. Furthermore, it shows that lentivirus-mediated CXCR4 transgene overexpression does not significantly alter the anti-catabolic and anti-inflammatory characteristics of CPCs.

[0161] In this discussion, FIG.7 shows: NF-kB and Erk activity is inhibited in response to SDF-1 stimulation in CPCs. Immunoblot analysis reveals that recombinant SDF-1 treatment reduces inhibitory kinase beta (IkB) levels in BM-MSCs (Left panel) indicating the NF-kB pathway activation. SDF-1 treatment increases IkB in hCPCs (Middle panel) and CXCR4-OE-hCPCs (Right panel). SDF-1 treatment also inhibits Erk pathways in both hCPCs and CXCR4-OE-hCPCs. Opposite results (activation of these pathways) are observed in BM-MSCs (Left panel). MAPK pathway was not significantly modulated upon SDF-1 treatment in BM-MSCs (Left panel), hCPCs (Middle panel) and CXCR4-hCPCs-OE (Right panel).

[0162] CXCR4-OE-hCPCs promote meniscus healing in rabbits: To investigate the therapeutic efficacy of CXCR4-OE-hCPCs in vivo, we analyzed the effect of these cells in a rabbit meniscus injury model (e.g., FIG.8). We created a 2.0 mm longitudinal tear that spans the entire thickness of the meniscus using a linear (non-circular, non-hollow) tissue punch. We then injected 5x106cells on Day 7 and again on Day 28, post-surgery. We split our injections into two, instead of just doing a single injection, to try to increase the time that cells would be presented into the injured knee. This was done in the event that the immune systems of these immunocompetent animals rapidly began clearing out the human cells.Attorney Docket No.: 405002-554001WO

[0163] At 60 days post-surgery, we performed microscopic histological assessment of their menisci for signs of healing at the tear site. Our results indicated that CXCR4-OE-hCPC-treated animals exhibited significant meniscal healing in comparison to the other two groups (FIG.2A). To evaluate the efficacy of each treatment, we quantified the number of animals in each experimental group that exhibited full meniscal healing / recovery at the time of harvest (FIG.2A, FIG.2B). The menisci of 83% of rabbits (5 of 6 animals) that received CXCR4-OE-hCPCs has healed, compared to only 50% (3 of 6) of the animals that received unmodified control CPCs and only 33% (2 of 6) of the saline control animals (FIG.2C). Additionally, we observed that the menisci from the CXCR4-OE-hCPCs exhibited higher proteoglycan content compared to the unmodified control hCPCs or saline controls as evidenced by the visibly robust Saf / O staining.

[0164] Fluorescence microscopy was used to closely inspect sections for the presence of human CPCs, which were fluorescently tagged. Interestingly, no fluorescent cells were observable in either of the cell-treated groups at 60 days post-injury, indicating that both CXCR4-OE-hCPCs and wild-type hCPCs had left the meniscus tissue by this time (not shown). This suggests that after stimulating the observed healing response, these human cells may have been cleared by the immune system of the host rabbit. We performed a secondary confirmation to ensure that the cells were truly absent in the tissue at this time using DNA analysis to detect human mitochondrial CYTB, which is only carried by human-origin cells. Results showed the absence of CYTB in the medial and lateral menisci as well as synovium (not shown).

[0165] Data are provided to support intraarticular injection of hCPCs-CXCR4-OE promotes meniscus healing in rabbits. FIG.8 shows an experimental setup and timeline of our in vivo study. Skeletally mature New Zealand White Rabbits were subjected to a longitudinal medial meniscal tear at Day 0. Animals were divided into three experimental groups based on the subsequent treatment that they received. Cell / saline control injections were administered on Day 7 and Day 28, post-surgery. Histopathology analysis of healing and joint health was performed 60 days post- surgery. FIG.2A shows representative histology images of medial menisci from rabbits following injury and treatment. The injury site is circumscribed. Menisci were sectioned and stained with Saf- O / Fast Green. Red staining signifies proteoglycan content. FIG.2C shows the percentage of animals with healed vs torn menisci in each of the three experimental groups. N = 6 animals per group.

[0166] CXCR4-OE-CPC treated animals exhibit significantly reduced cartilage erosion in the tibial plateau and femoral condyle: Joint histopathology analysis was used as a secondary outcome measure of treatment efficacy. The medial compartment of the tibial plateau and femoral condyle were assessed for signs of OA histopathology. Histological scoring was performed usingAttorney Docket No.: 405002-554001WO a modified OARSI scoring system. Tibial analysis revealed that animals treated with CXCR4-OE- hCPCs exhibited significantly improved cartilage histopathology in comparison to the saline control animals (FIG.2B). However, animals that received unmodified hCPC did not show significant improvement over the controls (FIG.2D). Assessment of histopathology scores of the femoral condyles showed the same trend. CXCR4-OE-hCPC-treated animals exhibited significantly improved scores compared to the saline control (FIG.2E, FIG.2F). Taken together, these results suggest that elevated CXCR4 expression is important for hCPCs to stimulate meniscus healing and prevent cartilage erosion in immunocompetent animals.

[0167] In FIG.2A and FIG.2B, animals with meniscal tears that were treated with intraarticular hCPCs-CXCR4-OE injection exhibit signs of attenuated PTOA. FIG.2B: Representative images of the medial tibial compartments of rabbits in each experimental treatment group. Sections were stained with Saf-O / Fast Green. Arrows signify articular cartilage lesion sites and / or visible and abrupt proteoglycan loss. FIG.2D OARSI histopathological scoring results are shown. FIG.2E: The medial side of the femoral condyles were also sectioned, stained, FIG.2F, and histologically scored. * p ≤ 0.05 indicates statistical significances between groups. N = 6 animals per group.

[0168] In the United States alone, meniscus injuries occur in 6 – 8% of active young adults annually. In high school athletes, for whom the most sophisticated epidemiological data exist, meniscus injuries account for 10 – 20% of all knee injuries1,10,13,15,20. Meniscal tears notoriously fail to heal on their own. This is most likely due to poor blood supply and hypocellularity of vulnerable areas like the white-white zone (WWZ)33,41. These injuries are often treated clinically by suturing and / or partial meniscectomy. However, they have a relatively high chance of reinjury as follow-ups at 86 months show a 19.1% failure rate35. The use of cell-based therapeutic strategies to enhance meniscus repair is an emerging area of research that has shown great promise. In a previous study, a progenitor cell line derived from healthy human cartilage (hCPCs) was developed, characterized, and its pre-clinical efficacy was validated in a rodent model. These cells were shown to endogenously produce less degradative proteinases such as MMP2, MMP13, ADMTS4, and ADMTS5, compared to than BM-MSCs,38which are the most commonly studied type of MSC for cell-based musculoskeletal tissue repair and regeneration. However, a limitation of the previous research on CPCs is that their in vivo efficacy for meniscus tear repair has only been demonstrated using a small animal model that is immunocompromised (RNU athymic rat model).

[0169] In the present study, we developed and tested an approach to maximize CPC efficacy for stimulating meniscus healing in a larger animal model that is not immunocompromised. Our main goal is to use this information to help predict the likelihood of success in human patientsAttorney Docket No.: 405002-554001WO and to acquire efficacy data that will facilitate the clinical translation of this innovative approach. Since the rabbit knee is appreciably larger than that of a rat, it was crucial that intraarticularly injected CPCs are actively recruited to the meniscal injury site, for maximizing healing. With this in mind, we aimed to promote their homing to the injury site. CXCR4 overexpression in stem cells has been reported to maximize cell migration, increase cell engraftment, and improve tissue regeneration in cell-based myocardial repair and ischemic tissue repair21,39,42. CXCR4 is a receptor for SDF-1, which is a chemokine that is secreted by meniscus tissues following acute injury, and it is crucial for the recruitment of satellite cells by injured tissues34. Unfortunately, culture expansion of mesenchymal lineage cells has been shown to significantly reduce endogenous CXCR4 expression (down to 5-8%)42.

[0170] To improve cell trafficking, we developed an hCPC line that constitutively overexpresses the CXCR4 gene (CXCR4-OE-hCPCs). We hypothesized that CXCR4 overexpression in CPCs would improve cell homing. Our initial characterization experiments confirmed that the generated CXCR4-OE-hCPCs have significantly higher expression of CXCR4 protein than unmodified / wild-type (WT) hCPCs. Cell survival assay results confirmed that lentiviral transduction and CXCR4 overexpression did not adversely affect their cell viability. Further, cell migration assays demonstrated that CXCR4-OE-hCPCs exhibit greater cell trafficking towards damaged meniscal fibrocartilage. To verify that this migratory response was indeed due to SDF-1 produced by the damaged meniscus tissue, we performed two separate experiments. In the first experiment, we quantified the amount of SDF-1 protein produced by damaged meniscal fibrocartilage. In the second experiment, we performed another cell migration assay, but instead of using damaged rabbit meniscus as the migratory target, we used a collagen sponge pre-soaked in a known quantity of recombinant SDF-1 protein. Veritably, our results showed that CXCR4-OE- hCPCs were migrating in response to rSDF-1 and that they did so with greater efficiency than WT- hCPCs.

[0171] In our final in vitro characterization experiment, we tested to see whether CXCR4 overexpression affects downstream SDF-1 pathway signaling. It has been previously demonstrated that CPCs outperform BM-MSCs in their stimulation of meniscus tissue regeneration and repair8,18. A unique characteristic of CPCs that may help to explain this outcome is that SDF- 1 treatment of these cells leads to significant inhibition of canonical NF-κB and Erk pathways, which are typically upregulated in response to SDF-1 in other cell types, including BM-MSCs27. To ensure that CXCR4 overexpression did not change this response to SDF-1, we examined the SDF- 1 / CXCR4 downstream signaling pathway in CXCR4-OE-hCPCs by analyzing the same downstream signaling molecules and found that they were largely consistent, with the exceptionAttorney Docket No.: 405002-554001WO that CXCR4-OE-hCPCs maintained a higher basal expression of protein IkB, which is a NF-κB pathway inhibitor. Overall, these findings are in agreement with previous observations that matrix metalloproteinase 13 (MMP13), a downstream target of NF-κB, is maintained at a lower expression level in CPCs than in BM-MSCs8,18.

[0172] Our evaluation of the therapeutic efficacy of CXCR4-OE-hCPCs revealed that intraarticular injections of these cells resulted in the complete healing of longitudinal meniscal tears in 83% of animals. These cells outperformed WT-hCPCs, which only resulted in complete healing in 50% of animals; and they far outperformed saline injection controls that saw healing in only 33%. Additionally, the menisci from the animals that received CXCR4-OE-hCPCs also exhibited visibly more proteoglycan retention, signified by richer Saf / O staining, as compared to animals in the other two groups. Histopathology scoring analysis of the medial compartment of the knee strongly suggested that the observed meniscal recovery in the animals that received CXCR4-OE-hCPCs also significantly improved the preservation of intact articular cartilage.

[0173] It was surprising that hCPCs were not detected in the menisci of rats at the 60-day harvest time point. Cells were injected on day 7 and day 28. Hence, the last cell injection occurred 32 days before harvest / analysis. This means that by the thirty-second day following injection, the cells had performed their intended function of accelerating meniscus healing and exited the tissue. From a regulatory perspective, it is important to recognize that the evacuation / clearance of these exogenous cells from the host knee, once meniscal healing has occurred, is indeed a major advantage of this approach. Still, we acknowledge that a clear limitation of our rabbit study is that it does not provide information about how long the injected human cells reside within the rabbit knee. The absence of detectable CPCs in the rabbit meniscus at the time of harvest and analysis suggests that these cells have left the knee or had been cleared out by the hosts’ immune system once their job was completed. One may even speculate that perhaps these human cells simply cannot survive the injection or the inflammatory microenvironment of the knee, altogether. To address this concern, we performed a proof-of-concept experiment to test whether human cells can indeed survive in the knees of immunocompetent animals after intraarticular injection (not shown). This validation experiment confirms that the fluorescently labeled human cells are detectable in Sprague Dawley rat meniscal sections following 72-hours post injection.

[0174] In summary, the present study demonstrates that elevating CXCR4 expression in human CPCs functionally increases their chemotactic migration toward targets that secrete SDF- 1. Further, our findings strongly suggest that administering these cells via intraarticular injection into the knee allows them to home in on sites of meniscal injury and ultimately result in meniscal healing and prevention of PTOA in immunocompetent animals (FIG.7). Future studies shouldAttorney Docket No.: 405002-554001WO focus on pinpointing the exact timing window of this cell-based repair response and determining its long-term efficacy in preventing OA development. In detail, FIG.7 shows CXCR4 overexpression and hCPC function. Collectively, our results suggest that increasing the expression of available CXCR4 on the cell surface of hCPCs makes them more sensitive / responsive to SDF- 1 ligand released by injured meniscus tissue. CXCR4 over expression increases the chemotaxis of these cells to the injured meniscus. SDF-1 uniquely attenuate NF-κB pathway activation, thereby inhibiting cellular catabolism and inflammatory signaling in hCPCs. In some embodiments, FIG.7 can be discussed as a provision of images acquired while measuring the protein expression of IkB- α and NF-kB p65. The Erk and MAPK pathway were analyzed by measuring Erk and p38 along with their phosphorylation states (pErk) and (p-p38). The data support SDF-1 inhibits inflammatory and catabolic pathways in CPCs. According to some aspects, the protein level expressions downstream targets of NF-kB pathway and Erk and MAPK pathway can be shown. In some embodiments, SDF-1 inhibits catabolic pathways in a dose dependent manner. FIG.11 shows an illustration diagram of CXCR4 overexpression and hCPC function including examples of increased chemotaxis, reduced catabolism, and reduced inflammation.

[0175] While contemplating additional details in the Detailed Description, and further adding various embodiments and examples, the following list of details can be inter-combined with any feature, aspect, and / or example disclosed herein:

[0176] Detail 1: A cell that accelerates musculoskeletal connective tissue repair, the cell comprising: a genetic modification such that the cell is a CXCR4 overexpressing (CXCR4 OE) cell; wherein the cell is a motile cell that can be administered to a subject in need thereof; and the cell accelerates musculoskeletal connective tissue repair compared to the same cell line that is not carrying the genetic modification.

[0177] Detail 2: The cell of detail 1, wherein the cell comprises a cartilage-derived progenitor cell line (CPC), a tenocyte, a chondrocyte, an adipocyte, an osteoblast, an osteocyte, an osteoclast, and / or a stem cell, and wherein the cell is derived from a tissue selected from the group consisting of cartilage, tendon, bone, adipose tissue, and muscle. Data is included herein (see Examples) for further support.

[0178] Detail 3: The cell of detail 1 or detail 2, wherein the cell comprises a low NF-Kb pathway activity in response to SDF-1 providing a suppressed catabolic activity, which is operative to affect cell-mediated anabolism in an inflammatory microenvironment, and wherein the low NF- Kb pathway activity is characterized by a reduced expression of one or more pro-inflammatory cytokines selected from the group consisting of IL-1, IL-6, and TNF-alpha.Attorney Docket No.: 405002-554001WO

[0179] Detail 4: The cell of detail 1, wherein the cell is included in a pharmaceutically acceptable formulation comprising a carrier, diluent, or excipient suitable for administration to a subject, and wherein the formulation is suitable for parenteral, intravenous, intramuscular, subcutaneous, or intra-articular administration.

[0180] Detail 5: The cell of detail 1, wherein the cell increases a tissue regeneration in the subject compared to an unmodified cell counterpart, and wherein the tissue regeneration comprises an increased synthesis of one or more extracellular matrix components selected from the group consisting of collagen, proteoglycans, and glycosaminoglycans.

[0181] Detail 6: A method of treating a subject diagnosed with a tissue injury or a subject suspected of having a tissue injury, the method comprising the steps of: (1) obtaining a pharmaceutical formulation comprising a plurality of cells comprising a genetic modification such that the cells are CXCR4 overexpressing (CXCR4 OE); and (2) administering a therapeutically effective amount of the cells via administering the pharmaceutical formulation; whereby a healing or repair of the tissue injury is accelerated compared to a treating using the same formulation without the CXCR4-OE.

[0182] Detail 7: The method of detail 6, wherein the cell comprises a cartilage-derived progenitor cell line (CPC), a tenocyte, a chondrocyte, an adipocyte, an osteoblast, an osteocyte, an osteoclast, and / or a stem cell, and wherein the cell is derived from a tissue selected from the group consisting of cartilage, tendon, bone, adipose tissue, and muscle.

[0183] Detail 8: The method of detail 6, wherein the cell comprises a low NF-Kb pathway activity in response to SDF-1 providing a suppressed catabolic activity, which is operative to affect cell-mediated anabolism in an inflammatory microenvironment, and wherein the low NF-Kb pathway activity is characterized by a reduced expression of one or more pro-inflammatory cytokines selected from the group consisting of IL-1, IL-6, and TNF-alpha.

[0184] Detail 9: The method of detail 6, wherein the cell is included in a pharmaceutically acceptable formulation comprising a carrier, diluent, or excipient suitable for administration to a subject, and wherein the formulation is suitable for parenteral, intravenous, intramuscular, subcutaneous, or intra-articular administration.

[0185] Detail 10: The method of detail 6, wherein the cell increases a tissue regeneration in the subject compared to an unmodified cell counterpart, and wherein the tissue regeneration comprises an increased synthesis of one or more extracellular matrix components selected from the group consisting of collagen, proteoglycans, and glycosaminoglycans.Attorney Docket No.: 405002-554001WO

[0186] Detail 11: The method of detail 6, wherein the pharmaceutical formulation is in the form of a suspension of the CXCR4 OE cells, and wherein the suspension comprises a physiologically compatible buffer or medium.

[0187] Detail 12: The method of detail 6, wherein the tissue comprises a connective tissue, a meniscus and / or a fibrocartilaginous tissue, and wherein the tissue injury is selected from the group consisting of a tear, a rupture, a partial tear, a partial rupture, and degeneration.

[0188] Detail 13: The method of detail 6, wherein the cell comprises a cartilage-derived progenitor cell line (CPC) isolated from a tissue selected from the group consisting of articular cartilage, auricular cartilage, costal cartilage, and nasal cartilage.

[0189] Detail 14: The method of detail 6, wherein the administering is via an intra-articular injection at or near an area of injury, and wherein the intra-articular injection is delivered to a joint selected from the group consisting of a knee joint, a hip joint, an ankle joint, a shoulder joint, an elbow joint, and a wrist joint.

[0190] Detail 15: A composition for accelerating musculoskeletal connective tissue repair, comprising a cell according to detail 1.

[0191] Detail 16: The composition of detail 15, wherein the cell comprises a cartilage- derived progenitor cell line (CPC), a tenocyte, a chondrocyte, an adipocyte, an osteoblast, an osteocyte, an osteoclast, and / or a stem cell, and wherein the cell is derived from a tissue selected from the group consisting of cartilage, tendon, bone, adipose tissue, and muscle.

[0192] Detail 17: The composition of detail 15, wherein the cell comprises a low NF-Kb pathway activity in response to SDF-1 providing a suppressed catabolic activity, which is operative to affect cell-mediated anabolism in an inflammatory microenvironment, and wherein the low NF- Kb pathway activity is characterized by a reduced expression of one or more pro-inflammatory cytokines selected from the group consisting of IL-1, IL-6, and TNF-alpha.

[0193] Detail 18: The composition of detail 15, wherein the cell is included in a pharmaceutically acceptable formulation comprising a carrier, diluent, or excipient suitable for administration to a subject, and wherein the formulation is suitable for parenteral, intravenous, intramuscular, subcutaneous, or intra-articular administration.

[0194] Detail 19: The composition of detail 15, wherein the cell increases a tissue regeneration in the subject compared to an unmodified cell counterpart, and wherein the tissue regeneration comprises an increased synthesis of one or more extracellular matrix components selected from the group consisting of collagen, proteoglycans, and glycosaminoglycans.Attorney Docket No.: 405002-554001WO

[0195] Detail 20: The composition of detail 15, further comprising a pharmaceutically acceptable carrier selected from the group consisting of a buffer, a diluent, a preservative, and a cryoprotectant.

[0196] Detail 21: The composition of detail 15, wherein the composition is formulated for parenteral administration, and wherein the parenteral administration is selected from the group consisting of intravenous, intramuscular, subcutaneous, and intra-articular administration.

[0197] Detail 22: A kit for accelerating a tissue repair in a subject in need thereof or in a subject suspected of having a tissue injury, the kit comprising: (1) a pharmaceutical formulation operative for accelerating a tissue repair, the pharmaceutical formulation including: a cell that is CXCR4-OE; and a solvent, powder, gas, or material that is capable of suspending the cell; and (2) a delivery device or configuration capable of delivering the suspended cell to a subject in need thereof.

[0198] Detail 23: The kit of detail 22, wherein the delivery device comprises a micro-needle or an injection needle operative to provide an intra-articular injection, or wherein the configuration comprises an injection without a needle, and wherein the intra-articular injection is delivered to a joint selected from the group consisting of a knee joint, a hip joint, an ankle joint, a shoulder joint, an elbow joint, and a wrist joint.

[0199] Detail 24: The cell of detail 1, the method of detail 6, or the composition of detail 15, wherein the cell is a CPC cell; and wherein a risk for a development of post-traumatic osteoarthritis (PTOA) is lowered by the cell, the method, and / or the kit compared to an administration of an unmodified CPC cell under the same conditions, and wherein the lowered risk for the development of PTOA is characterized by a reduced expression of one or more pro-inflammatory cytokines selected from the group consisting of IL-1, IL-6, and TNF-alpha.

[0200] Detail 25: The cell of detail 1, the method of detail 6, or the composition of detail 15, wherein SEQ ID NO: 1 is utilized for initialization in producing the CXCR4-OE and / or wherein an expression comprises SEQ ID NO: 2, and wherein the CXCR4-OE is characterized by an increased expression of CXCR4 mRNA and / or protein compared to an unmodified cell counterpart.

[0201] Detail 26: The cell of detail 25, the method of detail 25, or the composition of detail 25, wherein SEQ ID NO: 1 comprises: gtacaaaaaa gcaggctcca ccatggaggg gatcagtata tacacttcag ataactacac cgaggaaatg ggctcagggg actatgactc catgaaggaa ccctgtttcc gtgaagaaaa tgctaatttc aataaaatct tcctgcccac catctactcc atcatcttct taactggcat tgtgggcaat ggattggtca tcctggtcat gggttaccag aagaaactga gaagcatgac ggacaagtac aggctgcacc tgtcagtggc cgacctcctc tttgtcatca cgcttccctt ctgggcagtt gatgccgtgg caaactggta ctttgggaacAttorney Docket No.: 405002-554001WO ttcctatgca aggcagtcca tgtcatctac acagtcaacc tctacagcag tgtcctcatc ctggccttca tcagtctgga ccgctacctg gccatcgtcc acgccaccaa cagtcagagg ccaaggaagc tgttggctga aaaggtggtc tatgttggcg tctggatccc tgccctcctg ctgactattc ccgacttcat ctttgccaac gtcagtgagg cagatgacag atatatctgt gaccgcttct accccaatga cttgtgggtg gttgtgttcc agtttcagca catcatggtt ggccttatcc tgcctggtat tgtcatcctg tcctgctatt gcattatcat ctccaagctg tcacactcca agggccacca gaagcgcaag gccctcaaga ccacagtcat cctcatcctg gctttcttcg cctgttggct gccttactac attgggatca gcatcgactc cttcatcctc ctggaaatca tcaagcaagg gtgtgagttt gagaacactg tgcacaagtg gatttccatc accgaggccc tagctttctt ccactgttgt ctgaacccca tcctctatgc tttccttgga gccaaattta aaacctctgc ccagcacgca ctcacctctg tgagcagagg gtccagcctc aagatcctct ccaaaggaaa gcgaggtgga cattcatctg tttccactga gtctgagtct tcaagttttc actccagctg aatccaccca gctttcttgt ac, or (GenBank: EU831811.1, 1102 bp, DNA linear SYN, artificial sequence), and / or wherein SEQ ID NO: 1 encodes a CXCR4 protein.

[0202] Detail 27: The cell of detail 25, the method of detail 25, or the composition of detail 25, wherein SEQ ID NO: 2 comprises: megisiytsd nyteemgsgd ydsmkepcfr eenanfnkif lptiysiifl tgivgnglvi lvmgyqkklr smtdkyrlhl svadllfvit lpfwavdava nwyfgnflck avhviytvnl yssvlilafi sldrylaivh atnsqrprkl laekvvyvgv wipallltip dfifanvsea ddryicdrfy pndlwvvvfq fqhimvglil pgivilscyc iiisklshsk ghqkrkalkt tvililaffa cwlpyyigis idsfilleii kqgcefentv hkwisiteal affhcclnpi lyaflgakfk tsaqhaltsv srgsslkils kgkrgghssv stesesssfh ss, or (amino acid sequence, GenBank: ACE87329.1, artificial sequence, 352 AA), and / or wherein SEQ ID NO: 2 is a CXCR4 protein sequence.

[0203] Detail 28: A genetically modified CXCR4 overexpressing cell that accelerates tissue repair in a subject, the cell comprising: a genetic modification that causes the cell to overexpress CXCR4, wherein the genetic modification comprises introducing an exogenous nucleic acid encoding CXCR4 into the cell or activating an endogenous CXCR4 gene in the cell, wherein the cell is a motile cell selected from the group consisting of an adult stem cell, a mesenchymal stem cell, a hematopoietic stem cell, an induced pluripotent stem cell, an embryonic stem cell, a fibroblast, a keratinocyte, an endothelial cell, a cartilage-derived progenitor cell (CPC), a tenocyte, a chondrocyte, and an adipocyte, and wherein the cell is configured to be administered to the subject to accelerate tissue repair by shortening a time for the tissue repair compared to a time for tissue repair by an unmodified cell of the same cell line that lacks the genetic modification.Attorney Docket No.: 405002-554001WO

[0204] Detail 29: The cell of detail 28, wherein the cell is an immune cell selected from the group consisting of a T cell, B cell, macrophage, neutrophil, eosinophil, basophil, mast cell, and natural killer cell.

[0205] Detail 30: A method of accelerating tissue repair in a subject in need thereof, the method comprising: administering to the subject a genetically modified CXCR4 overexpressing cell at a site of tissue damage or systemically, wherein the genetically modified CXCR4 overexpressing cell comprises a genetic modification that causes the cell to overexpress CXCR4, the genetic modification comprising introducing an exogenous nucleic acid encoding CXCR4 into the cell or activating an endogenous CXCR4 gene in the cell, wherein the cell is a motile cell selected from the group consisting of an adult stem cell, a mesenchymal stem cell, a hematopoietic stem cell, an induced pluripotent stem cell, an embryonic stem cell, a fibroblast, a keratinocyte, an endothelial cell, a cartilage-derived progenitor cell (CPC), a tenocyte, a chondrocyte, and an adipocyte, wherein the genetically modified CXCR4 overexpressing cell accelerates the tissue repair by shortening a time for the tissue repair compared to a time for tissue repair by an unmodified cell of the same cell line that lacks the genetic modification, and wherein the tissue repair comprises wound healing, bone repair, cartilage repair, tendon repair, ligament repair, or skin repair.

[0206] Detail 31: The method of detail 30, wherein the genetically modified CXCR4 overexpressing cell is an immune cell selected from the group consisting of a T cell, B cell, macrophage, neutrophil, eosinophil, basophil, mast cell, and natural killer cell.

[0207] Detail 32: The method of detail 30, wherein the tissue is damaged tissue in the subject.

[0208] Detail 33: The method of detail 30, wherein administering the genetically modified CXCR4 overexpressing cell shortens the time for tissue repair by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to administering the unmodified cell.

[0209] Detail 34: The method of detail 30, wherein the unmodified cell is of the same cell type and from the same source as the genetically modified CXCR4 overexpressing cell but lacks the genetic modification.

[0210] Detail 35: The method of detail 30, wherein the exogenous nucleic acid encoding CXCR4 is operably linked to a promoter for expression in the cell.

[0211] Detail 36: The method of detail 30, wherein activating the endogenous CXCR4 gene comprises using a CRISPR / Cas9 system, a zinc finger nuclease system, a TALEN system, or a meganuclease system.

[0212] Detail 37: The method of detail 30, wherein the adult stem cell is obtained from bone marrow, adipose tissue, or peripheral blood of the subject or a donor.Attorney Docket No.: 405002-554001WO

[0213] Detail 38: The method of detail 31, wherein the immune cell is a T cell expressing a chimeric antigen receptor (CAR).

[0214] Detail 39: The method of detail 31, wherein the immune cell is a macrophage polarized to an M1 phenotype or an M2 phenotype.

[0215] Detail 40: The method of detail 30, wherein the fibroblast is a dermal fibroblast, the keratinocyte is an epidermal keratinocyte, and the endothelial cell is a vascular endothelial cell.

[0216] Detail 41: The method of detail 30, wherein the tissue repair comprises wound healing and the wound is a skin wound, a muscle wound, or an organ wound.

[0217] Detail 42: The method of detail 30, wherein administering the genetically modified CXCR4 overexpressing cell local to the site of tissue damage comprises injecting the cell into or adjacent to the damaged tissue.

[0218] Detail 43: The method of detail 30, wherein administering the genetically modified CXCR4 overexpressing cell systemically comprises intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0219] Detail 44: The method of detail 30, wherein the genetically modified CXCR4 overexpressing cell is administered in a composition further comprising a biocompatible material selected from the group consisting of a hydrogel, a polymer, a ceramic, and a decellularized tissue matrix.

[0220] Detail 45: The method of detail 44, wherein the biocompatible material is a porous scaffold or a hydrogel matrix.

[0221] Detail 46: The method of detail 30, wherein the subject is a mammal selected from the group consisting of a human, a mouse, a rat, a dog, a cat, a horse, a cow, a pig, and a non- human primate.

[0222] Detail 47: The method of detail 30, wherein the subject is a human patient in need of accelerated tissue repair.

[0223] Detail 48: The method of detail 30, further comprising administering one or more additional therapeutic agents that promote tissue repair.

[0224] Detail 49: The method of detail 48, wherein the one or more additional therapeutic agents are selected from the group consisting of growth factors, cytokines, antibodies, small molecules, and nucleic acids.

[0225] Detail 50: The method of detail 30, further comprising monitoring the tissue repair in the subject by imaging or assessing one or more biomarkers of tissue repair.Attorney Docket No.: 405002-554001WO

[0226] Detail 51: The method of detail 50, wherein the one or more biomarkers of tissue repair are selected from the group consisting of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), collagen, elastin, fibronectin, laminin, and glycosaminoglycans.

[0227] Detail 52: The method of detail 30, wherein the genetically modified CXCR4 overexpressing cell is autologous or allogeneic to the subject.

[0228] Detail 53: The method of detail 30, wherein the genetically modified CXCR4 overexpressing cell is administered in a single dose or in multiple doses over time.

[0229] Detail 54: The method of detail 30, wherein the genetically modified CXCR4 overexpressing cell is administered at a dose of about 1 x 10^4 to about 1 x 10^8 cells per administration.

[0230] The present disclosure can solve a long-felt but unmet problem that tissue repair takes a long time and the efficiency of tissue regeneration can be improved by administering a CXCR4 overexpressing (CXCR4 OE) cell to a subject; which can rapidly improve tissue regeneration in the subject. In an example, the technology provides a cell that accelerates a tissue repair in subject by shortening the amount of time for the tissue repair, the cell comprising a genetic modification such that the cell is a CXCR4 overexpressing (CXCR4 OE) cell; wherein the cell is a motile cell that can be administered to a subject in need thereof; and the cell accelerates the tissue repair by shortening the amount of time for the tissue repair compared to amount of time following an administration of the same cell line that is not carrying the genetic modification. While designing Examples, it was found that further example aspects that can be inter-combined with any detail, feature, aspect, and / or embodiment are as follows:

[0231] Example aspect 1: A cell that accelerates musculoskeletal connective tissue repair, the cell comprising: a genetic modification such that the cell is a CXCR4 overexpressing (CXCR4- OE) cell; wherein the cell is a motile cell that can be administered to a subject in need thereof; and the cell accelerates musculoskeletal connective tissue repair compared to the same cell line that is not carrying the genetic modification.

[0232] Example aspect 2: The cell of example aspect 1, wherein the cell comprises a cartilage-derived progenitor cell line (CPC).

[0233] Example aspect 3: The cell of example aspect 1 or example aspect 2, wherein the cell comprises a low NF-Kb pathway activity in response to SDF-1 providing a suppressed catabolic activity, which is operative to affect cell-mediated anabolism in an inflammatory microenvironment.

[0234] Example aspect 4: A pharmaceutical formulation comprising the cell of example aspect 1, example aspect 2, or example aspect 3.Attorney Docket No.: 405002-554001WO

[0235] Example aspect 5: A method of treating a subject diagnosed with a musculoskeletal connective tissue injury or a subject suspected of having a musculoskeletal connective tissue injury, the method comprising the steps of: (1) obtaining a pharmaceutical formulation comprising a plurality of cells comprising a genetic modification such that the cells are CXCR4 overexpressing (CXCR4-OE); and (2) administering a therapeutically effective amount of the cells via administering the pharmaceutical formulation; whereby a healing or repair of the musculoskeletal connective tissue injury is accelerated compared to a treating using the same formulation without the CXCR4- OE.

[0236] Example aspect 6: The method of example aspect 5 or the pharmaceutical formulation of example aspect 4, wherein the pharmaceutical formulation is in the form of a suspension of the cells.

[0237] Example aspect 7: The method of example aspect 5, wherein the connective tissue comprises a meniscus and / or a fibrocartilaginous tissue.

[0238] Example aspect 8: The method of example aspect 5, wherein the cell comprises a cartilage-derived progenitor cell line (CPC).

[0239] Example aspect 9: The method of example aspect 5, wherein the administering is via an intra-articular injection at or near an area of injury.

[0240] Example aspect 10: A kit for accelerating a musculoskeletal connective tissue repair in a subject in need thereof or in a subject suspected of having a musculoskeletal connective tissue injury, the kit comprising: (1) a pharmaceutical formulation operative for accelerating a musculoskeletal connective tissue repair, the pharmaceutical formulation including: a CPC cell that is CXCR4-OE; and a solvent, powder, gas, or material that is capable of suspending the cell; and (2) a delivery device or configuration capable of delivering the suspended cell to a subject in need thereof.

[0241] Example aspect 11: The kit of example aspect 10, wherein the delivery device comprises a micro-needle or an injection needle operative to provide an intra-articular injection, or wherein the configuration comprises an injection without a needle.

[0242] Example aspect 12: The cell of example aspect 1, the method of example aspect 5, or the kit of example aspect 10, wherein a risk for a development of post-traumatic osteoarthritis (PTOA) is lowered by the cell, the method, and / or the kit compared to an administration of an unmodified CPC cell under the same conditions.

[0243] Example aspect 13: The cell of example aspect 1, the method of example aspect 5, or the kit of example aspect 10, wherein SEQ ID NO: 1 is utilized for initialization in producing the CXCR4-OE and / or wherein an expression comprises SEQ ID NO: 2.Attorney Docket No.: 405002-554001WO

[0244] The disclosure provides a system for accelerating musculoskeletal connective tissue repair using a genetically modified cell. The core component of the system is a CXCR4 overexpressing (CXCR4 OE) cell, which may be a motile cell that can be administered to a subject in need. The genetic modification enables the cell to overexpress CXCR4, which may enhance the efficiency of tissue repair and shorten the amount of time required for tissue repair compared to an unmodified cell counterpart. The CXCR4 OE cell can be included in a pharmaceutically acceptable formulation, which may be administered therapeutically to a subject. The administration of the CXCR4 OE cell may increase tissue regeneration in the subject, providing an improvement over traditional methods. The system may also include a method for treating subjects diagnosed with or suspected of having a tissue injury by administering a therapeutically effective amount of the CXCR4 OE cells. The disclosure may encompass compositions, methods, and kits for accelerating tissue repair, highlighting the potential for improved outcomes in musculoskeletal connective tissue repair.

[0245] In the context of the system, the method may involve the administration of a CXCR4 overexpressing cell, which is a motile cell, to a subject in need. This administration may be aimed at accelerating musculoskeletal connective tissue repair. The CXCR4 OE cell may be genetically modified to overexpress the CXCR4 receptor, which may enhance its motility and therapeutic efficacy. The cell may be administered in a therapeutically effective amount, potentially increasing tissue regeneration in the subject compared to an unmodified cell counterpart. The administration may be performed via a pharmaceutical formulation, which may be in the form of a suspension of the CXCR4 OE cells. This formulation may be delivered through an intra-articular injection at or near the area of injury, providing a targeted therapeutic effect. The process may involve the utilization of SEQ ID NO: 1 for initialization in producing the CXCR4-OE, ensuring the genetic modification is achieved. The method may also include the specification of cell types, such as a cartilage-derived progenitor cell line (CPC), a tenocyte, a chondrocyte, an adipocyte, an osteoblast, an osteocyte, an osteoclast, and / or a stem cell, which may be used to tailor the treatment to the specific needs of the subject. The overall approach may aim to enhance tissue repair efficiency and shorten the time required for tissue repair, providing a therapeutic advantage.

[0246] According to an embodiment, the cell may be genetically modified to overexpress CXCR4, which may be achieved through the utilization of SEQ ID NO: 1 for initialization. This genetic modification may enable the cell to become a CXCR4 overexpressing (CXCR4 OE) cell. The CXCR4 OE cell may be administered to a subject in need, potentially providing a therapeutic effect. The administration of the CXCR4 OE cell may be intended to enhance tissue repair by shortening the time required for the repair process. The genetic modification may allow the cell toAttorney Docket No.: 405002-554001WO overexpress CXCR4, which may contribute to the acceleration of musculoskeletal connective tissue repair. The cell may be included in a pharmaceutically acceptable formulation, which may facilitate its administration. The formulation may be designed to ensure the stability and viability of the CXCR4 OE cells during administration. The CXCR4 OE cell may be administered in a therapeutically effective amount, which may be determined based on the specific needs of the subject. The administration may be performed via a suitable delivery method, such as an injection, to ensure the cells reach the target tissue. The CXCR4 OE cell may increase tissue regeneration in the subject compared to an unmodified cell counterpart, potentially leading to improved outcomes in tissue repair. The genetic modification and subsequent overexpression of CXCR4 may play a role in enhancing the cell's ability to repair tissue, making it a valuable tool in regenerative medicine.

[0247] The inclusion of a cell in a pharmaceutically acceptable formulation may be considered for the purpose of pharmaceutical formulation inclusion. The cell, which may be a CXCR4 overexpressing (CXCR4 OE) cell, can be included in a composition that is designed to accelerate musculoskeletal connective tissue repair. This inclusion may be achieved by formulating the cell in a manner that is pharmaceutically acceptable, ensuring that the cell can be administered to a subject in need thereof. The formulation may be designed to maintain the viability and functionality of the cell, allowing it to perform its intended function of accelerating tissue repair. The cell's genetic modification, which enables CXCR4 overexpression, may play a role in enhancing the cell's motility and its ability to accelerate tissue repair. The formulation may also be tailored to ensure that the cell can be effectively delivered to the target site, where it can exert its therapeutic effects. The potential for the cell to increase tissue regeneration compared to an unmodified cell counterpart may be a consideration in the formulation process. The formulation may be designed to optimize the cell's therapeutic potential, allowing it to contribute to the repair and regeneration of musculoskeletal connective tissue. The inclusion of the cell in a pharmaceutically acceptable formulation may thus be a step in the development of a composition that can effectively accelerate tissue repair in subjects in need thereof.

[0248] In the context of the system, the cell may comprise a low NF-Kb pathway activity in response to SDF-1, which may provide a suppressed catabolic activity. This suppressed catabolic activity may be operative to affect cell-mediated anabolism in an inflammatory microenvironment. The cell may be a CXCR4 overexpressing (CXCR4 OE) cell, which may be genetically modified to enable overexpression of CXCR4. This genetic modification may be carried by the cell, allowing it to be administered to a subject in need. The administration of the cell may be intended to provide treatment to a subject, potentially enhancing the efficiency of tissue repair and shortening theAttorney Docket No.: 405002-554001WO amount of time required for tissue repair. The cell may be included in a pharmaceutically acceptable formulation, which may facilitate its administration. The formulation may be designed to ensure the cell's viability and effectiveness upon delivery. The suppressed catabolic activity of the cell may contribute to its ability to accelerate musculoskeletal connective tissue repair and increase tissue regeneration in the subject compared to an unmodified cell counterpart. The overall process may involve the cell's interaction with the inflammatory microenvironment, where its low NF-Kb pathway activity may play a role in modulating the cellular response, potentially leading to improved tissue repair outcomes.

[0249] In the context of the system, the method, composition, and cell may comprise a cartilage-derived progenitor cell line (CPC), a tenocyte, a chondrocyte, an adipocyte, an osteoblast, an osteocyte, an osteoclast, and / or a stem cell. This specification of cell types may be integral to the system's operation. The inclusion of these specific cell types may facilitate the system's ability to address various aspects of tissue repair and regeneration. The method may involve the administration of a therapeutically effective amount of the cells, which may be administered to a subject in need. This administration may serve the function of providing treatment to a subject. The cells may be CXCR4 overexpressing (CXCR4 OE) cells, which may be motile and capable of accelerating tissue repair by shortening the amount of time required for the repair process. The genetic modification enabling CXCR4 overexpression may be a factor in enhancing the efficiency of tissue repair. The system may also include a pharmaceutically acceptable formulation, which may be in the form of a suspension of the CXCR4 OE cells. This formulation may be designed to facilitate the delivery and effectiveness of the cells in the treatment process. The potential for these actions to occur in a coordinated manner may suggest an approach to tissue repair, leveraging the specific cell types and genetic modifications to achieve the desired therapeutic outcomes.

[0250] In the context of the system, the pharmaceutical formulation may be prepared in the form of a suspension of CXCR4 overexpressing (CXCR4 OE) cells. This formulation may be designed to facilitate the administration of the cells to a subject in need. The suspension form may allow for a more efficient delivery of the therapeutic cells, potentially enhancing the treatment's effectiveness. The CXCR4 OE cells, characterized by their genetic modification, may be administered to a subject to accelerate tissue repair. This administration may involve delivering a therapeutically effective amount of the cells, which may be achieved through various methods, including intra-articular injection. The formulation as a suspension may provide a practical means of ensuring that the cells are delivered in a viable state, thereby maintaining their therapeutic potential. The process of administering the cells may be tailored to the specific needs of theAttorney Docket No.: 405002-554001WO subject, taking into account factors such as the location and severity of the tissue injury. The overall approach may aim to optimize the therapeutic outcomes by leveraging the unique properties of the CXCR4 OE cells, which may include enhanced motility and the ability to accelerate tissue repair.

[0251] In the context of a kit designed for tissue repair, a pharmaceutical formulation may be included, which is operative for accelerating tissue repair. This formulation may comprise a cell that is CXCR4 overexpressing (CXCR4 OE), which can be administered to a subject in need thereof. The kit may also include a solvent, powder, gas, or material capable of suspending the cell, facilitating its delivery. The delivery device or configuration may be capable of delivering the suspended cell to the subject, potentially through a micro-needle or an injection needle operative to provide an intra-articular injection. The kit may be designed to enhance the efficiency of tissue repair by utilizing the CXCR4 OE cell's ability to accelerate tissue repair, potentially shortening the amount of time required for the repair process. The inclusion of a delivery device may ensure that the cells are administered effectively to the target area, thereby optimizing the therapeutic administration. The kit's design may allow for flexibility in the choice of delivery method, accommodating various clinical needs and preferences.

[0252] In the context of the system, a delivery device may be utilized to facilitate the administration of a pharmaceutical formulation. This delivery device may comprise a micro-needle or an injection needle, which can be operative to provide an intra-articular injection. The intra- articular injection may be employed to deliver the formulation directly to the site of tissue injury, potentially enhancing the effectiveness of the treatment. The delivery device may be part of a kit that includes a pharmaceutical formulation operative for accelerating tissue repair. The formulation may contain CXCR4 overexpressing cells, which may be administered to a subject in need thereof. The administration of these cells may be intended to accelerate musculoskeletal connective tissue repair and increase tissue regeneration in the subject compared to an unmodified cell counterpart. The CXCR4 overexpressing cells may carry a genetic modification that enables overexpression of CXCR4, which may contribute to the enhanced tissue repair capabilities. The kit may be designed to provide a convenient and effective means of delivering the therapeutic cells to the target area, potentially improving the overall treatment outcome. The use of a micro-needle or injection needle for intra-articular injection may allow for precise delivery of the cells, which may be important for achieving the desired therapeutic effect. The formulation and delivery method may be tailored to meet specific treatment requirements, ensuring that the cells are delivered in a manner that maximizes their potential benefits.

[0253] In the context of the system, the method, composition, and cell may be associated with a potential reduction in the risk for the development of post-traumatic osteoarthritis (PTOA).Attorney Docket No.: 405002-554001WO This reduction may be facilitated by the administration of a CXCR4 overexpressing (CXCR4 OE) cell, which may be genetically modified to enhance its therapeutic efficacy. The CXCR4 OE cell may be administered to a subject in need, potentially providing a therapeutic effect that could lower the risk of PTOA compared to the administration of an unmodified cell counterpart. The cell may be included in a pharmaceutically acceptable formulation, which may be administered via a method that involves delivering a therapeutically effective amount of the cells to the subject. The administration may occur through an intra-articular injection, which may be facilitated by a delivery device such as a micro-needle or an injection needle. The formulation may be in the form of a suspension of the CXCR4 OE cells, which may enhance the delivery and efficacy of the treatment. The genetic modification of the cell may involve the utilization of SEQ ID NO: 1 for initialization in producing the CXCR4-OE, which may enable the overexpression of CXCR4 and contribute to the therapeutic potential of the cell. The cell may also comprise a low NF-Kb pathway activity in response to SDF-1, providing a suppressed catabolic activity that may be operative to affect cell- mediated anabolism in an inflammatory microenvironment. This suppressed catabolic activity may further contribute to the potential reduction in the risk of PTOA. The method, composition, and cell may be part of a kit that includes a pharmaceutical formulation operative for accelerating tissue repair, which may be delivered to a subject in need through a delivery device or configuration capable of providing an intra-articular injection. The kit may be designed to facilitate the administration of the CXCR4 OE cells, potentially enhancing the therapeutic outcome and reducing the risk of PTOA.

[0254] FIG. 12 is a flowchart illustrating a method in step 100 for obtaining a pharmaceutical formulation comprising a plurality of cells with a genetic modification, specifically CXCR4 overexpressing (CXCR4 OE) cells, according to an embodiment. At step 100, the pharmaceutical formulation may be obtained, which includes a plurality of cells that are genetically modified to overexpress CXCR4. This genetic modification may enable the cells to accelerate tissue repair by shortening the amount of time required for the repair process compared to cells that do not carry this modification. The cells may be motile, allowing them to be administered to a subject in need of tissue repair. The formulation may be designed to enhance the efficiency of tissue repair and to provide a therapeutic benefit to the subject. The cells within the formulation may also exhibit a low NF-Kb pathway activity in response to SDF-1, which may provide a suppressed catabolic activity, thereby affecting cell-mediated anabolism in an inflammatory microenvironment. The pharmaceutical formulation may be included in a pharmaceutically acceptable form, potentially as a suspension of the CXCR4 OE cells, to facilitate administration. The formulation may be administered via an intra-articular injection at or near the area of injury,Attorney Docket No.: 405002-554001WO targeting connective tissue, meniscus, or fibrocartilaginous tissue. The cells may comprise various types, including cartilage-derived progenitor cell lines (CPC), tenocytes, chondrocytes, adipocytes, osteoblasts, osteocytes, osteoclasts, and / or stem cells, to cater to different tissue repair needs. The overall process may aim to increase tissue regeneration in the subject compared to an unmodified cell counterpart, thereby potentially reducing the risk of developing post-traumatic osteoarthritis (PTOA).

[0255] In step 102 (FIG. 12), the process may involve administering a therapeutically effective amount of CXCR4 overexpressing cells through a pharmaceutical formulation. This administration may be intended to provide treatment to a subject in need, potentially enhancing the efficiency of tissue repair. The cells, which may include a variety of types such as cartilage- derived progenitor cells, tenocytes, chondrocytes, adipocytes, osteoblasts, osteocytes, osteoclasts, and stem cells, may be characterized by a genetic modification that enables the overexpression of CXCR4. This overexpression may contribute to the acceleration of tissue repair by shortening the time required for the repair process compared to cells that do not carry this genetic modification. The cells may also exhibit a low NF-Kb pathway activity in response to SDF- 1, which may provide a suppressed catabolic activity, thereby affecting cell-mediated anabolism in an inflammatory microenvironment. The pharmaceutical formulation may be in the form of a suspension of the CXCR4 OE cells, which may facilitate the administration process. The tissue targeted for repair may include connective tissue, meniscus, and / or fibrocartilaginous tissue. The administration may be performed via an intra-articular injection at or near the area of injury, which may further support the targeted delivery of the therapeutic cells to the site in need of repair. This approach may ultimately lead to an increase in tissue regeneration in the subject compared to an unmodified cell counterpart, thereby potentially enhancing the overall therapeutic outcome.

[0256] The particular embodiments disclosed above are illustrative only, as the application may be modified and practiced in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. It is therefore evident that the particular embodiments disclosed above may be altered or modified, and all such variations are considered within the scope and spirit of the application. Accordingly, the protection sought herein is as set forth in the description. It is apparent that an application with significant advantages has been described and illustrated. Although the present application is shown in a limited number of forms, it is not limited to just these forms but is amenable to various changes and modifications without departing from the spirit thereof.Attorney Docket No.: 405002-554001WO EXAMPLES EXAMPLE 1. PRODUCTION OF CXCR4-OE CPCS (CXCR4 OVEREXPRESSING CARTILAGE-DERIVED PROGENITOR CELL LINES)

[0257] In an example of cell culture, healthy (non-arthritic) human CPC cell lines were established as previously described in “Human Cartilage-Derived Progenitors Resist Terminal Differentiation and Require CXCR4 Activation to Successfully Bridge Meniscus Tissue Tears”E, Jayasuriya, CT. et al. Stem Cells, 37(1):102-114(2019), which is incorporated by reference herein in its entirety. For example, human tissue was obtained with approval from the Rhode Island Hospital Institutional Review Board. Healthy knee articular cartilage was obtained from an 18-year- old female patient undergoing an amputation. Healthy cartilage was also obtained from viable graft tissue from a 24-year-old male. Cartilage tissue was brought to the laboratory within the hour, washed thoroughly in 1× Hanks’ balanced saline solution (HBSS) and diced into very small pieces. The cartilage pieces were digested with Pronase (Roche, Indianapolis, IN; 2.0 mg / ml in 1× HBSS) for 30 minutes at 37°C in a shaking water bath. Cartilage pieces were washed in 1× HBSS and further digested using type IA Crude Bacterial Collagenase (Sigma–Aldrich, St. Louis, MO; 1.0 mg / ml) for 8 hours at 37°C in a shaking water bath. Cells were passed through a nylon cell strainer (100 μm pore size) to remove undigested tissue. Cells were washed three times in Dulbecco’s modified Eagle’s medium containing 10% (e.g., FIG.3).

[0258] In Immunoblotting, cells were lysed, and the protein lysate was resolved on polyacrylamide gel in equal concentration, which was then transferred onto a PVDF membrane, followed by blocking step. The proteins of interest were probed with respective antibodies, and the blots were then analyzed using ECL substrate.

[0259] For genetic modification, the hCPC cell line was modified further to overexpress the human CXCR4 gene (FIG.4A). The cells were also tagged with GFP for visualization and tracking. Control (unmodified) cells were generated for comparison, which contained a simple RFP gene tag in place of the human CXCR4 transgene gene construct (FIG.4B, FIG.4C). Western blot was used to confirm successful overexpression of CXCR4, compared to wild-type control cells as well as the RFP expressing control cells (FIG.4B, FIG.4C). An MTT assay was used to confirm that CXCR4 overexpression did not reduce cell viability (FIG.4D, FIG.4E). The “ns” (FIG.4E) refers to non-statistically significant differences between the indicated groups.

[0260] A suitable vector was obtained. FIG.5 shows the vector map for implementation; this is the expression construct that was delivered to CPCs by lentiviral delivery to generate CXCR4 OE CPCs. The lentivirus used to deliver the transgene construct: Manufacturer: DharmaconAttorney Docket No.: 405002-554001WO (Horizon) horizondiscovery.com / en; Variant: OHS5899-202624375; Item Name: Precision LentiORF CXCR4 w / Stop Codon; Clone Id: PLOHS_100066840. EXAMPLE 2. ANIMAL STUDIES

[0261] A medial parapatellar arthrotomy was performed on the right knees of skeletally mature New Zealand White rabbits. A 2.0 mm longitudinal bucket handle tear that spans the entire thickness of the meniscus was created. The patella was then returned to the natural position and the joint capsule, fascia, and skin were closed by suturing, respectively.5.0×106 cells were injected in respective groups at Day 7 and Day 28, post-op. Animals were divided in three experimental groups (6 animals / group): (1) CXCR4 overexpressing CPCs (CXCR4-OE CPC) injected; (2) Non targeting control CPCs (unmodified-CPCs) injected; (3) Saline injected controls. Animals were sacrificed 60 days post-op for analysis (e.g., FIG.2A, FIG.2B, FIG.2C, FIG.2D, FIG.2E, FIG.2F).

[0262] For histology analysis, the knee joint was harvested and fixed in 10% Neutral Buffered Formalin, followed by decalcification and processing. Samples were sectioned and stained with Safranin Orange (Saf-O) / Fastgreen. Modified double blinded OARSI Scoring was used to evaluate articular cartilage of tibial plateau.

[0263] Statistical analysis was was performed by Kruskal–Wallis test, which is an exemplary one-way analysis of variance.

[0264] The Examples and data above enable the technology, and such enablement has taken years of effort, various animal studies (e.g., rabbit / rat), and most often unpredictable results that have required repeated experimentation to achieve advancement. EXAMPLE 3. IN VITRO ASSAYS APPLICABLE TO IN VIVO

[0265] Materials and methods; Cell Culture: Healthy (non-arthritic) human CPC cell lines were established as described previously18. Briefly, non-arthritic human articular cartilage tissue was obtained with approval from Rhode Island Hospital, Providence, RI, USA. The cartilage tissue was washed thoroughly in 1× Hanks’ balanced saline solution (HBSS) (Gibco, USA) and was then diced into very small pieces followed by digestion using Pronase (Roche, USA) (2.0 mg / ml in 1× HBSS) and Collagenase (Sigma–Aldrich, USA) (1.0 mg / ml in 1× HBSS). Cells were then strained using a nylon cell strainer (100 μm pore size) and washed thrice in Dulbecco's modified Eagle's medium (DMEM) (GIBCO, USA) containing 10% fetal bovine serum (FBS) (GIBCO, USA). Enrichment of progenitor cells was achieved through fibronectin (FN) adhesion. After enrichment, the cells were immortalized using pRetro-E2 SV40 (Applied Biological Materials Inc., Canada), according to the manufacturer’s protocol. Cells were expanded and cultured in CPC-DMEM, which contains 10% FBS, 1% Pen Strep (GIBCO, USA), 100 mM HEPES (GIBCO, USA), 2 mM L-Attorney Docket No.: 405002-554001WO glutamine (GIBCO, USA), 0.1 mM ascorbic acid (SIGMA, USA), and 0.1 mM sodium pyruvate (GIBCO, USA) for further use.

[0266] To establish hCPCs constitutively overexpressing CXCR4, hCPCs were infected with lentivirus particles carrying Green Fluorescent Protein (GFP) downstream to the CXCR4 gene under the control of the human cytomegalovirus (HCMV) promotor according to the manufacturer’s protocol. The implemented non-targeting control vector was similar, but the CXCR4 transgene was replaced with a Red Fluorescent Protein (RFP) gene tag under the control of a HCMV promotor. The control vector contained a GFP gene tag as well, hence carrying dual fluorescence. The infection was followed by Puromycin (2mg / mL) (Sigma, USA) selection to eliminate uninfected cells. After the Puromycin selection, the pure population of infected cells was washed twice with 1× Phosphate Buffered Saline (PBS) to remove antibiotic residues. Cells were then cultured in CPC-DMEM and were subcultured regularly, at a density no less than 80% confluency, for future experiments. The overexpression of CXCR4 was confirmed by observation of Green fluorescence; and in the case of control cells, both Red and Green fluorescence, using a Nikon fluorescent microscope. Immunoblotting via Western blot analysis was used to validate the protein overexpression of CXCR4.

[0267] Immunoblotting: Cells were washed twice with 1× PBS to remove dead cells and remaining media. For cell lysis, RIPA lysis buffer (Cell Signaling Technology, USA) was then added to the cell monolayers and incubated on ice for 15-20 minutes. The lysate was then collected by centrifugation at 12,000 rpm for 20 minutes at 4°C to remove cell debris. The protein concentration in each sample was determined using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, USA), according to the manufacturer’s protocol. Cell lysates with the desired concentration were heat-denatured at 96°C for 5 minutes in the presence of 3× loading buffer (Cell Signaling Technologies, USA) containing 2 mM 1,4-Dithiothreitol (DTT). Samples were then loaded on the polyacrylamide gels and electrophoresis was performed at 100 V. The proteins were then transferred to an activated polyvinylidene fluoride (PVDF) membrane followed by blocking for 2 hours with 5% skimmed milk (Cell Signaling Technologies, USA) at room temperature. Blots were then washed thrice with 1× TBST containing 1% Twin 20. Blots were washed thrice with 1× TBST and then probed with primary antibodies against IkB (#4814), Erk (#4695), phospho-Erk (#4370), p38 (#9212) and phospho-p38 (#4511). β-Actin (#3700) was used as internal loading control. All the antibodies were purchased from Cell Signaling Technologies, USA. Blots with respective primary antibodies were incubated overnight at 4°C with gentle agitation. After incubation, blots were again washed thrice with 1× TBST and incubated with respective horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature. Two hours after the incubation,Attorney Docket No.: 405002-554001WO blots were washed and developed using a chemiluminescence substrate (Thermo Fischer Scientific, USA).

[0268] Cell survival assay: The survival / viability of the cells was determined using a VybrantTMMTT cell viability assay kit (Invitrogen, USA) according to the manufacturer’s protocol. Briefly, cells were seeded at the density of 20,000 cells per well in a 96-well plate containing 100 µL CPC-DMEM and incubated in a humidified CO2incubator at 37°C for 12 hours. Uninfected hCPCs were used as a negative control. Media was replaced with fresh 100 µL CPC-DMEM. Then, 10 µL of MTT (5 mg / mL) was added in each well in the dark and incubated at 37°C for the formation of formazan crystals. Four hours post-incubation, the crystals were dissolved using 100 µL dimethyl sulfoxide (DMSO) in each well and the absorbance was read at 570 nm.

[0269] Ex Vivo Cell Migration Assay: Fresh excised rabbit meniscal fibrocartilage was obtained and cut into small pieces (10 mg), and used for migration assay experiments. The damaged meniscal tissue (10 mg / well) was placed at the center of the 12-well plate and a 3.0- micron cell culture insert was placed to prevent the tissue from floating after the addition of CPC- DMEM. The 3.0-micron insert also allowed the free movement of small molecules and proteins (including SDF-1 released by the damaged tissue) across the membrane, but it prevented the cells from passing through. An 8.0-micron membrane insert was then placed on top and cells that wereseeded into the plate well at the density of 0.1×106cells / well. Cells were free to attach and migrateto the other side of the 8.0-micron membrane. The setup was then incubated in a humidified CO2incubator at 37°C. After 48 hours post-incubation, the 8-micron insert was removed and cells from the outer side of the membrane were collected using a cell scraper followed by the MTT cell proliferation assay to detect viable cells. Cells incubated without damaged menisci served as the negative controls.

[0270] A similar cell migration assay was performed using a collagen scaffold. CelluSponge (Advanced Biomatrix, USA) is a three-dimensional porous Collagen Type 1 coated HPC scaffold (Sigma–Aldrich, USA) that was pre-treated with SDF-1 recombinant protein (100 ng / mL) (PeproTech Inc., USA) for 8 hours at 4°C followed by similar steps as explained above.

[0271] Enzyme-Linked Immunosorbent Assay (ELISA): SDF-1 release in the supernatant was measured using ELISA kits according to the manufacturer’s protocols. EHCXCL12A (Thermo Scientific, USA) was used to determine the concentration of recombinant SDF-1 released from the Collagen Type 1 scaffold, in the experiment described above. EKC38542 (Biomatik, USA) was used to quantify SDF-1 released by injured rabbit tissue.

[0272] In vivo study; animal studies: All the animal studies were performed in accordance with approvals from the Institutional Animal Care and Use Committee of Rhode Island Hospital.Attorney Docket No.: 405002-554001WO Animals were anesthetized, randomized, and a medial parapatellar arthrotomy was performed on the right knees of male and female skeletally mature New Zealand White rabbits, followed by subluxation of the patella to access the medial meniscus. The meniscus was gently lifted off the tibia, with all attachments intact, and a 2.0 mm longitudinal tear penetrating the entire thickness of the meniscus (top to bottom) was created in red-white zone using a 2.0 mm hand surgery osteotome (ASSI, USA). We opted to use an osteotome instead of a scalpel blade to ensure controlled uniformity in all the dimensions of the injury. A smooth stainless-steel spatula was tucked between the meniscus and the medial tibial surface, to act as a backboard, during the creation of the injury. This ensured that the tibia was not damaged when pressure was applied on the osteotome to create the meniscal tear. The patella was then returned to natural position, and the joint capsule, fascia, and skin were closed by suturing, respectively. The animals were divided into three treatment groups (6 animals / group): (Group - 1) CXCR4 overexpressing CPCs (CXCR4-OE- hCPC) injection; (Group - 2) Non-targeting control CPCs (unmodified-control-hCPCs) injection; (Group -3) Saline control injection. A total of two injections (5.0×106cells each) were given. Cells were resuspended in 1× PBS. Injections were administered to each respective experimental group on Day 7 and Day 28, post-surgery. Animals were monitored and sacrificed humanely by euthanasia 60 days post-surgery for final tissue analysis.

[0273] Detection of Human DNA in Tissues: We used DNA analysis to detect the presence of human CPCs that were administered to rabbits. The presence of human DNA was independently checked in lateral menisci, medial menisci, and the synovium. This was done as previously described8,26. Briefly, primers were used to amplify the human mitochondrial cytochrome B gene (CYTB) by polymerase chain reaction (PCR). The amplicons were run using agarose gel electrophoresis to separate products based on size. Expected bands were observed under UVP Bio-Doc Transilluminator.

[0274] Histology Analysis: Knee joints were harvested and their medial menisci, femoral condyles, and tibial plateaus were collected. The tissues were then fixed in 10% Neutral Buffered Formalin followed by decalcification and processing. Samples were sectioned at 4 µm in the coronal plane for histological assessment of the meniscus and in the sagittal plane for articular cartilage. Menisci were stained with Safronin Orange / Fast Green and adjacent sister sections (collected approximately every 300 µm) were stained with DAPI. Tibial plateaus and femoral condyles were stained with Saf-O / Fast Green. Scoring of the tibial plateau and femoral condyle was conducted using Modified OARSI scoring23. Scoring took into account four different categorical criteria: (i) cartilage surface structure, (ii) Saf / O stain (iii) Cell density, and (iv) cluster formation. Histological assessment was performed using independent scoring by three seperate individualsAttorney Docket No.: 405002-554001WO who were blinded to the animal identifier and their respective surgical treatment group. The mean score for each experimental group was used for analysis.

[0275] Statistics: Statistical analysis was performed using one of two tests. For OARSI scoring analysis, a Kruskal–Wallis test, which is a non-parametric analysis of variance (ANOVA) on ranks, was implemented. For Cell Migration and MTT assay analyses, a Student’s T-test was used to compare experimental groups to their respective control group. N ≥ 3 for all experiments. Error bars represent one standard deviation (SD) of the mean. A p-value less than or equal to 0.05 was considered statistically significant.

[0276] To create hTNCL-CXCR4, the following protocols and mental exercises were executed: Thoughts were that tenocytes play a crucial role in the maintenance of tendon ECM. During the injury phase, tenocytes migrate to the injury site and proliferate to produce collagen and glycoproteins to promote healing, however, due to shorter retention at the injury site and slow proliferation rate, the repair is usually incomplete. Hence, we hypothesized that tenocytes constitutively overexpressing CXCR4 may result in their increased retention to the injury site and promote better healing. Primary Human Tenocytes were first obtained from patellar tendon from non-arthritic patients. Due to the limited availability of patient samples and the scarcity of primary tenocytes in tissue samples, we first created an immortalized cell line from human tenocytes (hTNCL) using lentiviral particles bearing hTERT downstream to the CMV promoter (FIG.9A). Infected cells were then subjected to Puromycin selection to eliminate uninfected cells and to obtain pure population of immortalized Tenocytes (hTNCL). hTNCL were then expanded in vitro, followed by cryopreservation for future use. hTNCL were then genetically modified using lentiviral vectors to constitutively overexpress CXCR4 transgene (hTNCL-CXCR4) using a third-generation lentivirus system (FIG.9B). For the infection controls, I replaced the CXCR4 gene with the Red Fluorescent Protein (RFP) gene, hence bearing dual fluorescence. The overexpression was confirmed by fluorescent imaging (FIG.9C). To further investigate the possible adverse effects of lentiviral infection and stable CXCR4 overexpression, I performed an MTT cell proliferation assay that indicates that neither cell immortalization nor CXCR4 overexpression has adverse effects on cells survival (FIG.9D).

[0277] In some embodiments, FIGs. 9A-9D illustrate establishment of CXCR4-hTNCL. (FIG.9A) Schematic representations of Human tenocyte immortalization (hTNCL) using CMV- hTERT lentiviral particles. (FIG.9B) Schematic representation of the establishment of hTNCL constitutively overexpressing CXCR4. (FIG.9C) Representative fluorescent microscopy images of control hTNCL (bearing dual reporter) and hTNCL-CXCR4. Images were taken at 10X magnification (FIG.9D) MTT cell proliferation assay (n=4) n.s. indicates not significant.Attorney Docket No.: 405002-554001WO

[0278] In representative experiments, it was found hTNCL-CXCR4 migrates in response to SDF-1 in vitro. Upon validation of CXCR4 overexpression and modulation of cell migration- related pathways, I performed an in vitro 3D cell migration assay using a collagen scaffold that was presoaked in different concentrations of SDF-1 (FIG.10A). The scaffolds were placed in the center of a well of 24 well plates and a 3-micron cell culture insert was placed to allow free migration of SDF-1 but will prevent the cell from migration. An 8-micron filter was placed on top of a 3-micron insert to allow transfer of cells. Cells were harvested from the outer side of the 8-micron insert and were quantified using an MTT assay. The results suggest increased migration of hTNCL-CXCR4 in response to SDF-1 in a dose-dependent manner. These findings collectively strengthen my hypothesis that hTNCL-CXCR4 will migrate toward injured patellar tendon tissue and will promote better healing (FIG.10B).

[0279] According to some aspects, FIGs.10A-10B illustrate hTNCL-CXCR4 migrates in response to SDF-1. (FIG.10A) Schematic representation of 3D cell migration assay. (FIG.10B) MTT assay to quantify the migrated cells from the outer side of the 8-micron cell culture insert. Wells without the scaffold were used as controls. *** = p value < 0.001.Attorney Docket No.: 405002-554001WO REFERENCES: A. Siegel L, Vandenakker-Albanese C, Siegel D. Anterior cruciate ligament injuries: anatomy, physiology, biomechanics, and management. Clin J Sport Med. 2012 Jul;22(4):349-55. doi: 10.1097 / JSM.0b013e3182580cd0. PMID: 22695402. B. Majewski M, Susanne H, Klaus S. Epidemiology of athletic knee injuries: a 10-year study. Knee 2006; 13:184–188. C. Boric-Persson F, Turkiewicz A, Neuman P, Englund M. Sick leave after arthroscopic meniscus repair vs. arthroscopic partial meniscectomy. Osteoarthr Cartil Open.2023 Jan 20;5(1):100340. doi: 10.1016 / j.ocarto.2023.100340. PMID: 36798736; PMCID: PMC9926294. D. 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J Knee Surg. 2019;32(2):123-126. 11. Englund M, Guermazi A, Lohmander SL. The role of the meniscus in knee osteoarthritis: a cause or consequence? Radiol Clin North Am.2009;47(4):703-712. 12. Englund M, Haugen IK, Guermazi A, et al. Evidence that meniscus damage may be a component of osteoarthritis: the Framingham study. Osteoarthritis Cartilage.2016;24(2):270-273. 13. Englund M, Roemer FW, Hayashi D, Crema MD, Guermazi A. Meniscus pathology, osteoarthritis and the treatment controversy. Nat Rev Rheumatol.2012;8(7):412-419. 14. Fox AJ, Wanivenhaus F, Burge AJ, Warren RF, Rodeo SA. The human meniscus: a review of anatomy, function, injury, and advances in treatment. Clin Anat.2015;28(2):269-287. 15. Gee SM, Tennent DJ, Cameron KL, Posner MA. The Burden of Meniscus Injury in Young and Physically Active Populations. Clin Sports Med.2020;39(1):13-27. 16. Jayasuriya CT, Chen Q. Potential benefits and limitations of utilizing chondroprogenitors in cell-based cartilage therapy. Connect Tissue Res.2015;56(4):265-271. 17. Jayasuriya CT, Chen Y, Liu W, Chen Q. The influence of tissue microenvironment on stem cell-based cartilage repair. Ann N Y Acad Sci.2016;1383(1):21-33. 18. Jayasuriya CT, Twomey-Kozak J, Newberry J, et al. Human Cartilage-Derived Progenitors Resist Terminal Differentiation and Require CXCR4 Activation to Successfully Bridge Meniscus Tissue Tears. Stem Cells.2019;37(1):102-114. 19. Jiang L, Lu J, Chen Y, et al. Mesenchymal stem cells: An efficient cell therapy for tendon repair (Review). Int J Mol Med.2023;52(2). 20. Jones JC, Burks R, Owens BD, et al. Incidence and risk factors associated with meniscal injuries among active-duty US military service members. J Athl Train.2012;47(1):67-73. 21. Kim M, Kim DI, Kim EK, Kim CW. CXCR4 Overexpression in Human Adipose Tissue- Derived Stem Cells Improves Homing and Engraftment in an Animal Limb Ischemia Model. Cell Transplant.2017;26(2):191-204. 22. Korpershoek JV, de Windt TS, Hagmeijer MH, Vonk LA, Saris DB. Cell-Based Meniscus Repair and Regeneration: At the Brink of Clinical Translation?: A Systematic Review of Preclinical Studies. Orthop J Sports Med.2017;5(2):2325967117690131. 23. Laverty S, Girard CA, Williams JM, Hunziker EB, Pritzker KP. The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the rabbit. Osteoarthritis Cartilage.2010;18 Suppl 3:S53-65.Attorney Docket No.: 405002-554001WO 24. Li J, Chen H, Zhang D, Xie J, Zhou X. The role of stromal cell-derived factor 1 on cartilage development and disease. Osteoarthritis Cartilage.2021;29(3):313-322. 25. Li X, Li D, Li J, et al. Preclinical Studies and Clinical Trials on Cell-Based Treatments for Meniscus Regeneration. Tissue Eng Part B Rev.2023;29(6):634-670. 26. Matsuda H, Seo Y, Kakizaki E, et al. Identification of DNA of human origin based on amplification of human-specific mitochondrial cytochrome b region. Forensic Sci Int.2005;152(2- 3):109-114. 27. McCorkell KA, May MJ. Noncanonical NF-κB activation and SDF-1 expression in human endothelial cells. Methods Mol Biol.2015;1280:155-180. 28. Mueller MB, Tuan RS. Functional characterization of hypertrophy in chondrogenesis of human mesenchymal stem cells. Arthritis Rheum.2008;58(5):1377-1388. 29. Newberry J, Desai S, Adler C, et al. SDF-1 preconditioned HPC scaffolds mobilize cartilage-derived progenitors and stimulate meniscal fibrocartilage repair in human explant tissue culture. Connect Tissue Res.2020;61(3-4):338-348. 30. Niu W, Guo W, Han S, et al. Cell-Based Strategies for Meniscus Tissue Engineering. Stem Cells Int.2016;2016:4717184. 31. Petit I, Jin D, Rafii S. The SDF-1-CXCR4 signaling pathway: a molecular hub modulating neo-angiogenesis. Trends Immunol.2007;28(7):299-307. 32. Rai MF, Brophy RH, Sandell LJ. Osteoarthritis following meniscus and ligament injury: insights from translational studies and animal models. Curr Opin Rheumatol.2019;31(1):70-79. 33. Raj MA, Bubnis MA. Knee Meniscal Tears. In: StatPearls. Treasure Island (FL): StatPearls Publishing Copyright © 2024, StatPearls Publishing LLC.; 2024. 34. Saito Y, Shimada M, Utsunomiya T, et al. Homing effect of adipose-derived stem cells to the injured liver: the shift of stromal cell-derived factor 1 expressions. J Hepatobiliary Pancreat Sci. 2014;21(12):873-880. 35. Schweizer C, Hanreich C, Tscholl PM, et al. Nineteen percent of meniscus repairs are being revised and failures frequently occur after the second postoperative year: a systematic review and meta-analysis with a minimum follow-up of 5 years. Knee Surg Sports Traumatol Arthrosc.2022;30(7):2267-2276. 36. Shen W, Chen J, Zhu T, et al. Intra-articular injection of human meniscus stem / progenitor cells promotes meniscus regeneration and ameliorates osteoarthritis through stromal cell-derived factor-1 / CXCR4-mediated homing. Stem Cells Transl Med.2014;3(3):387-394.Attorney Docket No.: 405002-554001WO 37. Somoza RA, Welter JF, Correa D, Caplan AI. Chondrogenic differentiation of mesenchymal stem cells: challenges and unfulfilled expectations. Tissue Eng Part B Rev.2014;20(6):596-608. 38. Twomey-Kozak J, Desai S, Liu W, et al. Distal-Less Homeobox 5 Is a Therapeutic Target for Attenuating Hypertrophy and Apoptosis of Mesenchymal Progenitor Cells. Int J Mol Sci. 2020;21(14). 39. Wang Y, Sun X, Lv J, et al. Stromal Cell-Derived Factor-1 Accelerates Cartilage Defect Repairing by Recruiting Bone Marrow Mesenchymal Stem Cells and Promoting Chondrogenic Differentiation. Tissue Eng Part A.2017;23(19-20):1160-1168. 40. Ward RJ, Driban JB, MacKay JW, et al. Meniscal degeneration is prognostic of destabilzing meniscal tear and accelerated knee osteoarthritis: Data from the Osteoarthritis Initiative. J Orthop Res.2023;41(11):2418-2423. 41. Yan W, Dai W, Cheng J, et al. Advances in the Mechanisms Affecting Meniscal Avascular Zone Repair and Therapies. Front Cell Dev Biol.2021;9:758217. 42. Yang JX, Zhang N, Wang HW, et al. CXCR4 receptor overexpression in mesenchymal stem cells facilitates treatment of acute lung injury in rats. J Biol Chem.2015;290(4):1994-2006. 43. Yu H, Adesida AB, Jomha NM. Meniscus repair using mesenchymal stem cells - a comprehensive review. Stem Cell Res Ther.2015;6(1):86.

Claims

Attorney Docket No.: 405002-554001WO CLAIMS We claim:

1. A cell that accelerates musculoskeletal connective tissue repair, the cell comprising: a genetic modification such that the cell is a CXCR4 overexpressing (CXCR4 OE) cell; wherein the cell is a motile cell that can be administered to a subject in need thereof; and the cell accelerates musculoskeletal connective tissue repair compared to the same cell line that is not carrying the genetic modification.

2. The cell of claim 1, wherein the cell comprises any cell that comes from connective tissue, such as (but not limited to) a cartilage-derived progenitor cell line (CPC), a tenocyte, a chondrocyte, an adipocyte, an osteoblast, an osteocyte, an osteoclast, a synovial cell, a meniscal cell, and / or a mesenchymal stem cell.

3. The cell of claim 1 or claim 2, wherein the cell comprises a low NF-Kb pathway activity in response to SDF-1 providing a suppressed catabolic activity, which is operative to affect cell- mediated anabolism in an inflammatory microenvironment; and wherein a low NF-Kb pathway is such that the NF-Kb pathway of the cell is lower than that, all other things being equal, of a cell without the genetic modification of claim 1.

4. The cell of claim 1, wherein the cell is included in a pharmaceutically acceptable formulation.

5. The cell of claim 1, wherein the cell increases tissue regeneration in the subject compared to an unmodified cell counterpart.

6. A method of treating a subject diagnosed with a tissue injury or a subject suspected of having a tissue injury, the method comprising the steps of: (1) obtaining a pharmaceutical formulation comprising a plurality of cells comprising a genetic modification such that the cells are CXCR4 overexpressing (CXCR4-OE); and (2) administering a therapeutically effective amount of the cells via administering the pharmaceutical formulation; whereby a healing or repair of the tissue injury is accelerated compared to a treating using the same formulation without the CXCR4-OE.

7. The method of claim 6, wherein the cell comprises any cell that comes from connectiveAttorney Docket No.: 405002-554001WO tissue, such as (but not limited to) a cartilage-derived progenitor cell line (CPC), a tenocyte, a chondrocyte, an adipocyte, an osteoblast, an osteocyte, an osteoclast, a synovial cell, a meniscal cell, and / or a mesenchymal stem cell.

8. The method of claim 6, wherein the cell comprises a low NF-Kb pathway activity in response to SDF-1 providing a suppressed catabolic activity, which is operative to affect cell-mediated anabolism in an inflammatory microenvironment.

9. The method of claim 6, wherein the cell is included in a pharmaceutically acceptable formulation.

10. The method of claim 6, wherein the cell increases a tissue regeneration in the subject compared to an unmodified cell counterpart.

11. The method of claim 6, wherein the pharmaceutical formulation is in the form of a suspension of the CXCR4 OE cells.

12. The method of claim 6, wherein the tissue comprises a connective tissue, a meniscus and / or a fibrocartilaginous tissue and / or wherein the cell includes a cartilage-derived progenitor cell line (CPC), a tenocyte, a chondrocyte, an adipocyte, an osteoblast, an osteocyte, an osteoclast, a synovial cell, a meniscal cell, and / or a mesenchymal stem cell.

13. The method of claim 6, wherein the cell comprises a cartilage-derived progenitor cell line (CPC), a tenocyte, a chondrocyte, an adipocyte, an osteoblast, an osteocyte, an osteoclast, a synovial cell, a meniscal cell, and / or a mesenchymal stem cell.

14. The method of claim 6, wherein the administering is via an intra-articular injection at or near an area of injury, and / or wherein the administering includes a superficial injection into tendon and / or ligament.

15. A composition for accelerating musculoskeletal connective tissue repair, comprising a cell according to claim 1.

16. The composition of claim 15, wherein the cell comprises any cell that comes fromAttorney Docket No.: 405002-554001WO connective tissue, such as (but not limited to) a cartilage-derived progenitor cell line (CPC), a tenocyte, a chondrocyte, an adipocyte, an osteoblast, an osteocyte, an osteoclast, a synovial cell, a meniscal cell, and / or a mesenchymal stem cell.

17. The composition of claim 15, wherein the cell comprises a low NF-Kb pathway activity in response to SDF-1 providing a suppressed catabolic activity, which is operative to affect cell- mediated anabolism in an inflammatory microenvironment.

18. The composition of claim 15, wherein the cell is included in a pharmaceutically acceptable formulation.

19. The composition of claim 15, wherein the cell increases a tissue regeneration in the subject compared to an unmodified cell counterpart.

20. The composition of claim 15, further comprising a pharmaceutically acceptable carrier.

21. The composition of claim 15, wherein the composition is formulated for parenteral administration.

22. A kit for accelerating a tissue repair in a subject in need thereof or in a subject suspected of having a tissue injury, the kit comprising: (1) a pharmaceutical formulation operative for accelerating a tissue repair, the pharmaceutical formulation including: a cell that is CXCR4-OE; and a solvent, powder, gas, or material that is capable of suspending the cell; and (2) a delivery device or configuration capable of delivering the suspended cell to a subject in need thereof.

23. The kit of claim 22, wherein the delivery device comprises a micro-needle or an injection needle operative to provide an intra-articular injection; and / or wherein the configuration comprises an injection without a needle; and / or wherein the administering includes a superficial injection into tendon and / or ligament or into an end of a tendon and / or ligament.Attorney Docket No.: 405002-554001WO 24. The cell of claim 1, the method of claim 6, or the composition of claim 15, wherein the cell is a CPC cell; and wherein a risk for a development of post-traumatic osteoarthritis (PTOA) is lowered by the cell, the method, and / or the kit compared to an administration of an unmodified CPC cell under the same conditions.

25. The cell of claim 1, the method of claim 6, or the composition of claim 15, wherein the cell, the method, or the composition is included in application including a use of one or more CXCR4 OE tenocytes for a tendinopathy.

26. The cell of claim 1, the method of claim 6, or the composition of claim 15, wherein SEQ ID NO: 1 is utilized for initialization in producing the CXCR4-OE and / or wherein an expression comprises SEQ ID NO:

2.

27. The cell of claim 1, the method of claim 6, or the composition of claim 15, wherein SEQ ID NO: 1 comprises: gtacaaaaaa gcaggctcca ccatggaggg gatcagtata tacacttcag ataactacac cgaggaaatg ggctcagggg actatgactc catgaaggaa ccctgtttcc gtgaagaaaa tgctaatttc aataaaatct tcctgcccac catctactcc atcatcttct taactggcat tgtgggcaat ggattggtca tcctggtcat gggttaccag aagaaactga gaagcatgac ggacaagtac aggctgcacc tgtcagtggc cgacctcctc tttgtcatca cgcttccctt ctgggcagtt gatgccgtgg caaactggta ctttgggaac ttcctatgca aggcagtcca tgtcatctac acagtcaacc tctacagcag tgtcctcatc ctggccttca tcagtctgga ccgctacctg gccatcgtcc acgccaccaa cagtcagagg ccaaggaagc tgttggctga aaaggtggtc tatgttggcg tctggatccc tgccctcctg ctgactattc ccgacttcat ctttgccaac gtcagtgagg cagatgacag atatatctgt gaccgcttct accccaatga cttgtgggtg gttgtgttcc agtttcagca catcatggtt ggccttatcc tgcctggtat tgtcatcctg tcctgctatt gcattatcat ctccaagctg tcacactcca agggccacca gaagcgcaag gccctcaaga ccacagtcat cctcatcctg gctttcttcg cctgttggct gccttactac attgggatca gcatcgactc cttcatcctc ctggaaatca tcaagcaagg gtgtgagttt gagaacactg tgcacaagtg gatttccatc accgaggccc tagctttctt ccactgttgt ctgaacccca tcctctatgc tttccttgga gccaaattta aaacctctgc ccagcacgca ctcacctctg tgagcagagg gtccagcctc aagatcctct ccaaaggaaa gcgaggtgga cattcatctg tttccactga gtctgagtct tcaagttttc actccagctg aatccaccca gctttcttgt ac, or (GenBank: EU831811.1, 1102 bp, DNA linear SYN,Attorney Docket No.: 405002-554001WO artificial sequence).

28. The cell of claim 1, the method of claim 6, or the composition of claim 15, wherein SEQ ID NO: 2 comprises: megisiytsd nyteemgsgd ydsmkepcfr eenanfnkif lptiysiifl tgivgnglvi lvmgyqkklr smtdkyrlhl svadllfvit lpfwavdava nwyfgnflck avhviytvnl yssvlilafi sldrylaivh atnsqrprkl laekvvyvgv wipallltip dfifanvsea ddryicdrfy pndlwvvvfq fqhimvglil pgivilscyc iiisklshsk ghqkrkalkt tvililaffa cwlpyyigis idsfilleii kqgcefentv hkwisiteal affhcclnpi lyaflgakfk tsaqhaltsv srgsslkils kgkrgghssv stesesssfh ss, or (amino acid sequence, GenBank: ACE87329.1, artificial sequence, 352 AA).

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