EP4093425A1 - Polyhedrin delivery system releasing growth factors - Google Patents

Abstract

A composition is provided comprising a protein complex with growth factors that are particularly useful for treating musculoskeletal diseases and cartilage damage, especially osteoarthritis. A delivery system is also provided to enable sustained release of the growth factors.

Description

POLYHEDRIN DELIVERY SYSTEM RELEASING GROWTH FACTORS

FIELD OF THE INVENTION

The present invention relates to compositions and methods for the treatment of cartilage diseases or injuries. In particular, the present invention relates to treatment of cartilage defects and osteoarthritis in a subject. The invention can be used in research, medical applications and veterinary applications.

BACKGROUND

Musculoskeletal disorders, diseases and injury can be extremely debilitating. Osteoarthritis (OA) is a type of arthritis typified by cartilage degradation, synovial inflammation and changes to subchondral bone. It is a progressive degenerative disease and approximately 8.75 million people in the UK aged 45 years and over have sought treatment for OA, with symptoms of joint stiffness, impaired mobility and persistent pain, all of which contribute to a diminished work capacity and quality of life(1 ).

There are currently no available disease modifying treatments for OA. Standard treatments include pain management, anti-inflammatory medication, lifestyle changes and regenerative medicine strategies. As the symptoms increase and non-surgical therapies are no longer effective, joint replacement surgery is the standard intervention.

OA can be caused by joint surface defects and one experimental approach to treat these in recent years has been to use cellular scaffold implants to promote cartilage and bone growth. These scaffolds can be made from a range of materials, including ex vivo collagen preparations, hydrogels, polymers and ceramics.

Often the scaffolds are populated with cells and/or substances such as growth factors. Bone morphogenetic proteins (BMPs) are a class of growth factor which are known to upregulate chondrocyte proliferation, stimulate cartilage growth, promote recruitment of chondroprogenitors, and up-regulate synthesis of extracellular matrix (ECM) components including collagen fibres and proteoglycans. They have also been known to exhibit chondroprotective activity in vivo (2-6). One such scaffold is disclosed in WO 2012/096997 which is an implantable metal/polymer scaffold for synovial joint repair. WO2018/215752 discloses implantable hydrogel scaffolds for tissue repair such as bone or cartilage in conditions such as osteoarthritis. US8916228B2 discloses biomedical scaffolds with porous layers for use in treating tissue defects. The microchannels allow ingress and habitation of cells and growth factors to promote bone regeneration in the area surrounding the scaffold. US6911212B2 discloses a malleable bone putty designed to be retained at the site of repair and not be washed away by blood or fluids brought to the site by the healing mechanism. The putty can be impregnated with materials such as TGF-beta, PDGF, BMPs, IGF-1 , antibiotics and living cells. Whereas, US20180256507A1 discloses corticosteroid loaded microparticles for reducing inflammation or pain in a joint. The core shell structure of the microparticles allows for sustained highly localised release of the drug.

The primary problem associated with these types of scaffolds and applications is premature protein denaturation and uncontrolled burst release of substances from the scaffold and/or microparticle (7). Scaffold deformation and uncontrolled burst release is also more likely in highly moveable joints, such as synovial joints. Consequently, high doses of substances such as growth factors which coat the scaffold or microparticles are required to achieve efficacy, which can cause toxicity and side effects such as excessive and/or off-target ossification/bone growth. The increased amount of growth factors required also renders these types of interventions expensive. The use of standard recombinant BMPs as therapeutic agents is also limited, in particular due to their fragility, with half-lives ranging between minutes and hours. This prevents a prolonged exposure to therapeutic levels of growth factors. Incorporation into scaffolds such as polylactide-co-glycolide (PLGA) has been limited due to protein denaturation and burst release. Consequently, high doses are required to achieve efficacy, which can lead to toxicity and off-target side effects (8).

The present invention seeks to address some of these problems.

SUMMARY OF THE INVENTION

According to the present invention there is provided a composition comprising a polyhedrin protein in combination with at least one growth factor. Preferably, the polyhedrin protein complex forms a crystal scaffold. The growth factors can be incorporated within the polyhedrin complex.

The growth factor may be selected from OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11 , BMP-12, BMP-13, BMP-15, BMP- 16, BMP-17. BMP-18, DPP, FGF-2, FGF-18, Vgl, Vgr, 60A protein, GDF-1 , GDF-2, GDF- 3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9,GDF-10, GDF-11 , GDF-12,CDMP-1 , CDMP-2, CDMP-3, NODAL, UNIVIN, SCREW, ADMP, NEURAL, IGF-1 , and amino acid sequence variants thereof.

By way of example, the composition may comprise a polyhedrin protein together with one or more bone morphogenic protein (BMP). The composition may additionally comprise any other therapeutic agents suitable for the disease to be treated and that can be formulated in the polyhedrin protein complex.

By way of example, the composition may comprise a polyhedrin protein and BMP-7. Alternatively, the composition may comprise polyhedrin protein and BMP-2. In a further embodiment the composition may comprise polyhedrin protein, BMP-7 and BMP-2.

In another aspect of the invention there is provided the use of a composition comprising a polyhedrin protein in combination with at least one growth factor in the treatment of musculoskeletal conditions, disorders, diseases or injury in a subject.

The musculoskeletal condition can be any condition sustained by a mammal such as an injury of the cartilage,

The composition can be used to treat any diseases where treatment requires the use of growth factors. In particular diseases such as osteoarthritis, where cartilage is damaged.

The treatment may involve promoting sustained cellular proliferation and/or production of extracellular matrix at the site of administration. For example, growth factors used in the compositions of the invention once delivered to the site of disease enhance the cellular proliferation and production of the extracellular matrix in the region of osteoarthritis. The compositions or the invention enable an increased production of ECM components such as GAGs, hyaluronic acid, fibronectin, or chondroitin sulphate, and, upregulation in the expression of ECM genes such as COLIA1 , COL2A1 and ACAN mRNA in chondrocytes, promoting chondrogenesis, ECM synthesis, cell proliferation and healing of chondral defects at the site of administration.

The subject to be treated may be selected from any mammal such as humans, horses, dogs, cats and livestock, amongst others where treatment with growth factors is required.

In accordance with another aspect of the invention there is provided a pharmaceutical formulation comprising a composition with a polyhedrin protein in combination with at least one growth factor and an evaluated pharmaceutically acceptable carrier selected. The pharmaceutically acceptable carrier can be selected from a physiological solution comprising any one or a combination of glucose, dextrose, normal saline, phosphate buffered saline (PBS) or Ringer's solution, or any other suitable carrier.

Preferably the polyhedrin forms a crystalline scaffold complex with the growth factors comprised within the scaffolds in the formulation.

The pharmaceutical formulation will be optimised from the candidate formulations, namely, in a gel, hydrogel, tablet, capsule, liquid, injectable solution, suspension or powder.

In a further aspect of the invention there is provided a delivery system comprising a polyhedrin protein in combination with at least one growth factor where the growth factor is released from the polyhedrin at the site of administration in a sustained manner, in a delayed manner or in a gradient-like manner.

The composition is preferably delivered at the site of musculoskeletal injury, or at the site of a disease such as in the cases of cartilage defect or damage. For joint disease the composition and/or formulation would be delivered via intra-articular injection as directed by the results of in-vivo studies. In particular, the present invention can be used to treat cartilage defects and osteoarthritis. The treatment may involve promoting sustained cellular proliferation at the site of administration, of, for example, chondrocytes, chondroprogenitors, mesenchymal stem or stromal cells, and synovial cells.

The treatment may involve one or more of increased glycosaminoglycan (GAG) production, upregulation in the expression of ECM component genes such as COL2A1 and ACAN in chondrocytes, promoting chondrogenesis, ECM synthesis, and chondrocyte proliferation to repair chondral defects directly at or surrounding the site of administration. This occurs due to the release of bioactive PODS^® -incorporated growth factor into the surrounding environment, leading to the activation of signalling pathways to promote cartilage formation or repair.

Bone morphogenetic proteins (BMPs), such as BMP-2 and BMP-7, have been implicated in cartilage homeostasis and repair, and are promising OA disease modifying candidates. Several studies have demonstrated that these BMPs stimulate an anabolic response in cartilage explants and articular chondrocytes, promote recruitment of chondroprogenitors, and up-regulate synthesis of ECM components including collagen fibres and proteoglycans. Furthermore, BMP-2 and BMP-7 have been shown to be chondroprotective in small and large animal models of OA (2-6).

The Polyhedrin Delivery System (PODS®) (Cell Guidance Systems Ltd, UK) is a protein manufacturing platform technology which has been developed to overcome limitations of the known scaffolds and delivery systems. Viral polyhedar complexes and their methods of use are disclosed in WO 2008/105672A1 , WO 2004/063371 A1 and WO 2002/36785A1 .

PODS® technology harnesses polyhedrin, a component of the Bombyx mori cypovirus infectious lifecycle, where the synthesised polyhedrin protein produces a complex, highly organised crystal scaffold in which virions are constrained to protect them following release to the external environment (9). This survival mechanism allows for a longer window of opportunity for the embedded virions to be ingested. Once ingested, the virions can reach target intestinal cells intact, where cargo is released enabling viral transmission. This mechanism has been adapted to create PODS® proteins, which are synthesised within insect cells by co-expression of polyhedrin and a cargo protein which is incorporated via an immobilization tag which binds the polyhedrin protein (10). Constraint of a growth factor within a crystalline form allows for sustained release of functional, biologically active growth factor. Degradation of PODS®is mediated by cell and protease dependent mechanisms, enabling release of cargo protein at physiologically relevant levels over a period of weeks to months, from a single application of crystals as demonstrated in WO 2018/189501 , where a growth factor gradient was provided by the PODS®system.

The inventors have shown, for the first time, that the polyhedrin system comprising BMP-2 and BMP-7 promotes cartilage repair and can be used to treat diseases such as OA.

The inventors have also found that even low levels of PODS®crystals containing growth factors such as BMPs exert an enhanced, prolonged therapeutic effect on chondrocyte proliferation, ECM synthesis and cartilage repair in vitro and in vivo. In vitro, low levels of PODS®crystals resulted from the periodic removal of cellular media, thereby removing half of the growth factor released from the PODS®each time, yet a significant effect on ECM production was still observed.

The present invention is particularly useful in the treatment of cartilage-associated disorders such as osteoarthritis.

Preferably, the methods and compositions are used for the treatment of humans or for veterinary use, such as for the treatment of animals with cartilage-associated conditions, including in mammals such as horses, canines, dogs, cats and livestock.

The term "cartilage disorder" or "cartilage-associated disorder" as used herein refers to a medical condition that includes as a characteristic, reduced or damaged cartilage as compared to a control or normal subject. A cartilage disorder may result from disease or injury and/or reduced cartilage. The cartilage-associated disorder may be osteoarthritis.

The term "pharmaceutically acceptable carrier" as used herein refers to the acceptance or use of the carrier in the pharmaceutical industry. Preferably the carrier is approved for use in humans. Exemplary carriers include physiological solutions including but not limited to glucose, dextrose, normal saline, phosphate buffered saline (PBS) or Ringer's solution. The term "therapeutically effective amount" as used herein refers to an amount of an active ingredient that produces the intended result.

The term “crystalline formulation” as used herein refers to an ordered crystalline protein lattice consisting of polyhedrin protein.

Crystalline formulation in combination with hydrogel, aqueous solution, paste, liquid, other biomaterials such as electrospun fabrics Combinations may be prepared with any of the following: OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11 , BMP-12, BMP-13, BMP-15, BMP-16, BMP-17. BMP-18, DPP, FGF-2, FGF-18, Vgl, Vgr, 60A protein, GDF-1 , GDF-2, GDF- 3, GDF- 5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11 , GDF-12,CDMP-1 , CDMP-2, CDMP-3, NODAL, UNIVIN, SCREW, ADMP, NEURAL, IGF-1 , and amino acid sequence variants thereof.

Dosage regimens may consist of either multiple injections over a long period of time (e.g., administration once per month), or a single injection. Dosage of PODS® would vary depending on the species (due to differences in joint cavity volume and size), and could vary between 0.01 ng/kg to 500 mg/kg, depending on the PODS® growth factor of interest. Each integer within that range is exemplified as preferred embodiments.

FIGURES

The invention will now be described by way of Examples and Figures as an illustration only, in which:-

Figure 1 (A to B): shows graphs illustrating the effect of PODS® BMP-2 (pBMP-2) on real-time primary chondrocyte proliferation. Cell proliferation was monitored in real time using the xCELLigence E-plate, which converts cell impedance to cell index. Chondrocytes were cultured up to 14 days with a range of pBMP-2 (25-200 ng/ml equivalent), alongside PODS® Empty (pEmpty) (200 ng/ml equivalent), and recombinant BMP-2 (100 ng/ml). No media changes were performed over the entire culture period. Cell index is normalised to the initial cell seeding peak at 4 hours in Figure 1 A and Day 4 in Figure 1 B, respectively. Traces represent the mean cell index for each treatment group.

Figure 2 (A to B): shows a graph illustrating the effect of PODS® BMP-7 (pBMP-7) on real-time primary chondrocyte proliferation. Cell proliferation was monitored in real- time using the xCELLigence E-plate, which converts cell impedance to cell index. Chondrocytes were cultured up to 14 days with a range of pBMP-7 (25-200 ng/ml equivalent), alongside pEmpty (200 ng/ml equivalent), and recombinant BMP-7 (200 ng/ml). No media changes were performed over the entire culture period. Cell index is normalised to the initial cell seeding peak at four hours in Figure 2A and Day 4 in Figure 2B. Traces represent the mean cell index for each treatment group.

Figure 3 (A to B): shows photographs with Alcian blue staining of chondrocytes. Alcian blue staining was performed to measure GAG content after primary chondrocytes were cultured in micromass over 21 days. Figure 3A shows representative staining after culturing with pBMP-2 or pBMP-7 (both 50 ng/ml equivalent), alongside pEmpty (50 ng/ml equivalent), standard rBMP-2 (100 ng/ml) or standard rBMP-7 (200 ng/ml). Figure 3B shows representative staining after culturing with a dose range of pBMP-2 or pBMP-7 (25-150 ng/ml equivalent). A half media change was performed every 3-4 days during the culture period, where standard recombinant growth factor was replenished while no additional crystals were added to PODS® treated wells.

Figure 4 (A to C): shows the influence of PODS® growth factors on ECM mRNA expression. qRT-PCR was performed to measure ECM marker expression in chondrocytes cultured in micromass over 7 days. Figure 4A shows the relative expression when cells were cultured with pBMP-2 or pBMP-7 (both 50 ng/ml equivalent), alongside pEmpty (50 ng/ml equivalent), standard rBMP-2 (100 ng/ml) or standard rBMP-7 (200 ng/ml). Figures 4B and 4C illustrates the relative expression of ECM markers when chondrocytes were cultured with a dose range of pBMP-2 or pBMP-7 (25-100 ng/ml equivalent), respectively. A half media change was performed on Day 3 of the culture period, where standard recombinant growth factor was replenished while no additional PODS® crystals were added to PODS® treated wells. Points and error bars represent the mean and standard deviation.

Figure 5: shows the repair of cartilage defect in murine model after intra-articular injection of PODS® crystals. Safranin-0 staining of sections from injured joints of 8- week old C57BL/6 mice. A longitudinal, full-thickness cartilage defect was created in the mice with a 27G needle, after which mice were intra-articularly injected with the treatment (all n=5 per group). Mice sacrificed at four weeks post-surgery were treated with 18.75 ng equivalent pBMP-2, pBMP-7, or pEmpty in PBS or 0.5% hydrogel, or 0.5% hydrogel alone. Mice sacrificed at eight weeks post-surgery were treated with 18.75 ng equivalent pBMP-2, pBMP-7 or pEmpty in 0.5% hydrogel. Joints were fixed, sectioned and stained with Safranin O for histological analysis of cartilage repair. Images show representative repair of cartilage for each treatment group at 4 or 8 weeks post-surgery.

Figure 6 (A to D): shows modified Pineda and Mankin histology scoring of cartilage repair and OA in murine model after intra-articular injection with PODS® crystals. Safranin-0 staining of histological sections from injured joints of 8-week old C57BL/6 mice were assessed for cartilage repair and OA using the Modified Pineda and Mankin scoring systems. A longitudinal, full-thickness cartilage defect was created in the mice with a 27G needle, after which mice were intra-articularly injected with the treatment (all n=5 per group). Mice sacrificed at four weeks post-surgery were treated with pBMP-2, pBMP-7, or pEmpty in PBS or 0.5% hydrogel, or 0.5% hydrogel alone. Mice sacrificed at eight weeks post-surgery were treated with pBMP-2, pBMP-7 or pEmpty in 0.5% hydrogel. 6.75x105 PODS® crystals were administered per treatment group, equivalent to 18.75 ng of protein. Joints were fixed, sectioned and stained with Safranin O for histological analysis of cartilage repair at 4 and 8 weeks. Sections were scored by two observers blinded to the group assignment to assess the level of cartilage repair and osteoarthritis. Bars show the mean score with error bars representing the standard error and ^** indicates significant (p<0.05) improvement in histological score compared with Week 4 pooled data.

EXAMPLES

The proliferation of chondrocytes and production of cartilage ECM in vitro were first investigated to confirm efficacy of PODS® compared with standard recombinant growth factor, and to provide guidance on dosage in vivo. Administration of PODS® by intra-articular injection into the knee joint was performed with a composition of PODS® BMP-2 (pBMP-2) and PODS® BMP-7 (pBMP-7) which demonstrated healing of a chondral defect in an in vivo murine model of cartilage repair.

Example 1 - Synthesis of PODS® BMP-2 and PODS® BMP-7 pBMP-2 and pBMP-7 were synthesised as previously described(11) using standard methods. Both constructs contained full-length BMP-2 and BMP-7 protein (NCBI accession numbers P12643 and P18075, respectively) and were fused to the H1 incorporation tag as described in US8554493B1 .

Transfer DNA was co-transfected into Spodoptera frugiperda 9 (Sf9) cells with linearised baculovirus (BV) DNA using TranslT®-lnsect (Mirus Bio). Replication- competent BV was rescued by recombination between the transfer vector and linearised viral DNA. Virus was harvested and plaque purification performed to isolate a single recombinant BV. Plaques were screened and BV was harvested to infect Sf9 cells to produce PODS® crystals. Subsequently, crystals were harvested and purified by lysing Sf9 cells using successive rounds of sonication and PBS washes. Purified PODS® were sterility tested and lyophilised prior to use in experiments. Although equivalence depends on context, 3.6x104 pBMP-2 or pBMP-7 crystals is approximately functionally equivalent to 1 ng of standard rBMP-2 or rBMP-7, respectively, in terms of bioactivity (12). The dosage of PODS® is based on crystal number as the amount of active growth factor incorporated into the crystal is unknown. This way it enables the reader to know how many crystals were used when we refer to PODS® in ng/ml, to put the crystal number of PODS® into a ‘dosage’ context’. The sustained release effect of PODS® enables a lower dose of growth factor to be administered, resulting in a cheaper treatment, less toxicity and therefore fewer off- target side effects.

Example 2 - Primary chondrocyte isolation

Primary human articular chondrocytes were isolated from cartilage obtained from donors undergoing total knee arthroplasty for osteoarthritis. Tissue was collected under ethical consent obtained from the Local Ethics committee and the UK Home Office (Project Licence number 70/8635). Articular cartilage was shaved from the subchondral bone, cut into slices using a sterile scalpel, and washed in PBS. Chondrocytes were isolated from the cartilage by further mincing the cartilage slices and incubating them with digestion buffer at 37°C, 5% 02 on a shaking platform (55 rpm). Digestion buffer consisted of Dulbecco’s Modified Eagle medium (DMEM) (1 g/l glucose) with GlutaMAX™, pyruvate and Phenol Red (ThermoFisher Scientific) supplemented with 10% foetal bovine serum (FBS) (First Link UK), 1x penicillin/streptomycin (P/S) (ThermoFisher Scientific), and 6 mg/ml collagenase A (Sigma Aldrich). After digestion, the cell suspension was filtered through a 70 pm MACS SmartStrainer (Miltenyi Biotec), centrifuged at 300xg and washed with PBS. Isolated primary chondrocytes were resuspended and expanded in growth media (DMEM (1 g/l glucose) with GlutaMAX™, pyruvate and phenol red supplemented with 10% FBS and 1xP/S).

Example 3 - Cell Culture

Chondrocytes were cultured up to passage 3 in hypoxia (3% 02) using a HeraCell™ Vios Tri-Gas Incubator (Fisher Scientific).

Clonetics™ Normal Human Articular Chondrocytes (NHAC-kn) isolated from a six- year old female (lot number 6F4018, Lonza) were expanded in monolayer in DMEM (1 g/l glucose) with GlutaMAX™, pyruvate and phenol red (ThermoFisher Scientific) supplemented with 10% foetal bovine serum (FBS) (First Link UK) and 1x penicillin- streptomycin-glutamine (PSG). Following the manufacturer’s guidelines, NHAC-kn cells were cultured up to passage 15. Phagocytosis of PODS® crystals were observed within 48 h of addition.

To promote ECM production, primary OA chondrocytes and NHACs were seeded as a micromass of 2x 105 cells (10 mI/well). After incubating for two hours at 37°C, 5% C02, 250 pi of media was slowly added. Cells were treated with standard rBMP-2 (100 ng/ml) (Cell Guidance Systems), standard rBMP-7 (200 ng/ml) (ThermoFisher Scientific), PODS® Empty (pEmpty) (50 ng/ml) (Cell Guidance Systems), pBMP-2 (25-200 ng/ml) (Cell Guidance Systems), or pBMP-7 (25-200 ng/ml) (Cell Guidance Systems). A half media change was performed every 3-4 days for PODS® containing wells (with no addition or replacement of crystals), or a half media change with replacement of growth factor for wells containing standard rBMP-2 or rBMP-7.

Example 4 - qRT-PCR

Total cellular RNA was isolated from cells after 7 days of micromass culture using the RNeasy® Mini Kit (Qiagen), following the manufacturer’s protocol, including an on- column gDNA elimination treatment (Qiagen). RNA concentration and quality was quantified using UV-spectrometry with the Nanodrop ND-2000 Spectrophotometer (ThermoFisher Scientific) and stored at -80°C. Reverse transcription was performed using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s instructions and cDNA stored at -20°C. qRT-PCR was performed using 2x Fast SYBR Green (ThermoFisher Scientific) with commercial primers for the following human genes: collagen typel (COL1A1), aggrecan (ACAN), and hypoxanthine-guanine phosphoribosyltransferase (HPRT) and b-actin (BACT) (housekeeping genes) (all obtained from ThermoFisher Scientific). The collagen type II (COL2) forward and reverse primers sequences were 5'- TG G GTGTTCT ATTT ATTT ATTGTCTTCCT -3 ' (SEQ ID NO: 1 ) and 5'- GCGTTGGACTCACACCAGTTAGT-3' (SEQ ID NO: 2) respectively (Integrated DNA Technologies). PCR conditions were 95°C for 20 seconds, followed by 40 cycles of 95°C for 3 seconds and 60°C for 30 seconds.

Example 5 - Alcian blue

Alcian blue staining was assessed after 21 days of micromass culture. Cells were washed with PBS and fixed in 10% formalin for twenty minutes. After fixing, cells were washed with PBS before the addition of 1% Alcian blue (Sigma) diluted in 0.1 M HCI. This was incubated overnight at room temperature, after which cells were washed three times with 0.1 M HCI, then washed with distilled water. Alcian blue staining was visualised by bright microscopy.

Example 6 - Real-time cell proliferation

Real-time monitoring of cell proliferation was performed using the xCELLigence® RTCA DP instrument (ACEA Biosciences Inc.). Primary OA chondrocytes or NHAC- kn cells were serum starved overnight in DMEM + 1% FBS before treatment with standard rBMP-2 (100 ng/ml), standard rBMP-7 (200 ng/ml), pEmpty (200 ng/ml equivalent, pBMP-2 (25-200 ng/ml), or pBMP-7 (25-200 ng/ml). The xCELLigence E- plate wells (ACEA Biosciences Inc.) were filled with 50 pi of DMEM + 1% FBS and incubated at room temperature for 2 hours. Cells were then seeded at 1 x 103 cells per well and incubated at room temperature for 30 minutes. Proliferation was monitored for up to 14 days without any media changes or further supplements.

Example 7 - In vivo murine model of cartilage regeneration

In this study we assessed the intra-articular response to PODS® crystals and their suitability for use as an OA therapy. We created a chondral lesion in the patella groove as previously described by Eltawil et al. 2009 (13). All procedures were approved by the Local Ethics committee and the UK Home Office (Project Licence number 70/8635). Animals were used to assess the reaction of the intra-articular environment to the introduction of PODS®(with/without growth factors and in the presence/absence of a carrier hydrogel), and to assess the effect on the healing of the chondral defect after four and eight weeks.

Eight-week old female C57/BL6 mice were given subcutaneous 1 mg/kg buprenorphine as a pre-operative analgesia before being anaesthetised by 4% isoflurane, which was then maintained at 1 .5-2% during the procedure. Medial para patellar arthrotomy was performed using a dissection microscope, by placing the animal in a dorsal recumbent position. The knee joint was flexed and the joint capsule opened by blunt dissection and the patella luxated laterally to expose the patella groove articular surface. A longitudinal full thickness injury to the cartilage defect was created with a sterilised 27G needle down the trochlea sulcus. The joint was injected with 1 pi of each treatment (n=5 mice per group) shown in Table 1 , using a 2mI Hamilton syringe (Sigma Aldrich). 6.75x105 PODS® crystals (equivalent to 18 ng of protein) were administered for each treatment group either in PBS or in 0.5% (v/v) peptide- based hydrogel (PuraStat® [3D Matrix Medical Technology]), with pEmpty and 0.5% hydrogel alone serving as negative control groups. After creation of the defect, the patella was repositioned and sutured (Ethicon). The contra-lateral knee was left non- operated.

Table 1 - Murine model treatment groups

Example 8 - Histology Histological analysis was performed at Week 4 to assess the response of the joint to the administration of intra-articular PODS® and at Week 8 post-surgery to assess the rate of joint healing and OA using industry-standard scoring methods. Animals were humanely sacrificed at four and eight weeks post-surgery and stifles retrieved. Knee joints were fixed and decalcified in 10% EDTA pH 8 for 14 days. The joints were processed through a series of sequential ethanol and xylene immersions with the Leica TP1020 Semi-enclosed Benchtop Tissue Processor (Leica Biosystems) and paraffin embedded using the HistoCore Arcadia (Leica Biosystems). Joints were serially sectioned at 7 pm intervals using the Leica Biosystems RM2245 Semi- Automated Rotary Microtome (Leica Biosystems). Three sections per animal were scored from the middle of the defect.

Sections were stained with Safranin O. After deparaffinisation and rehydration, sections were stained with Weigert’s iron haematoxylin (Sigma Aldrich) working solution for 10 minutes, followed by washing under tap water for 10 minutes. Slides were transferred to 0.1% Fast Green FCF (Sigma Aldrich) for 5 minutes, before being transferred to 1% acetic acid for 10 to 15 seconds. Subsequently slides were stained in 0.1% Safranin O solution for 5 minutes. Sections were then dehydrated through 100% ethanol, cleared with xylene and mounted using DPX mounting medium. Slides were analysed with a Nikon Eclipse Ti inverted Microscope, and images captured with an Orca OSG camera (Nikon, Japan) using NIS-Elements Advanced Research software.

Joint surface repair of the chondral defect was scored using the Modified Pineda scoring system (Table 2), and OA in the joint was scored using the Mankin histology scoring system (Table 3). The entire joint was sectioned and three slides per animal were scored from the middle of the lesion. Scoring was performed independently by two observers blinded to the group assignment.

Table 2 - Modified Pineda histology scoring system(14) Table 3 - Mankin histology scoring system(15,16)

Example 9 - PODS® BMP-2 and PODS® BMP-7 stimulate chondrocyte proliferation.

The xCELLigence assay was used to monitor real-time changes in cellular proliferation in the presence of pBMP-2 (Figures 1 and 2) or pBMP-7 (Figures 3 and 4) compared to their respective standard recombinant counterparts. This instrument measured changes in cell impedance (caused by changes in the total cell surface area in contact with the bottom of the well) to generate a cell index, from which cell number is inferred. Chondrocytes were cultured with between 9x105-7.2x106 pBMP-2 or pBMP-7 (equivalent to 25-200 ng/ml standard recombinant growth factor), to assess whether there is a dose-dependent effect of PODS® on proliferation. This was compared to standard rBMP-2 (100 ng/ml), rBMP-7 (200 ng/ml), and pEmpty (25 or 200 ng/ml equivalent). Chondrocytes were cultured up to 14 days with no media change over the entire period. Cell index was normalised to the initial cell seeding peak at 4 hours (Figures 1 A and 2A) or Day 4 (Figure 1 B and 2B) to assess whether PODS® induces sustained proliferation.

Over the first two days, standard rBMP-2 or rBMP-7 generated a sharp increase in proliferation, with an increase in normalised cell index up to 2.7 and 1 .7, respectively. The effect of PODS® on chondrocyte proliferation was similar for both pBMP-2 and pBMP-7. For these groups, as well as for pEmpty, there was a slight increase in normalised cell index over this period, with a higher peak for the lower numbers of PODS® which may be related to initial post-seeding changes in cell impedance. After the first two days of culture, there was a decline in normalised cell index for all treatment groups up to Day 4.

Between Day 4 and Day 14, with higher concentrations of pBMP-2 and pBMP-7 (100, 150 and 200 ng/ml equivalent) there was a steady increase in normalised cell index by Day 14, with a peak average normalised cell index of 1 .8-2.7 for pBMP-2, and 2.2- 4.6 for pBMP-7. This augmentation in normalised cell index was more marked for the two highest doses of PODS®. By contrast, there was no increase in normalised cell index between Days 4 and 14 for rBMP-2, rBMP-7, pEmpty, and the lower concentrations of pBMP-2 or pBMP-7 (25 ng/ml and 50 ng/ml). This suggests that a single application of pBMP-2 and pBMP-7 promotes cellular proliferation in a dose- dependent manner over a sustained period of two weeks, an effect which is not reproducible with pEmpty or standard recombinant growth factor.

Example 10 - PODS® BMP-2 and PODS® BMP-7 induce ECM genes and proteoglycan synthesis

Chondrocytes were cultured with pBMP-2, pBMP-7, pEmpty (all 50 ng/ml equivalent), standard rBMP-2 (100 ng/ml), or standard rBMP-7 (200 ng/ml). GAG production was assessed after 21 days of micromass culture using Alcian blue staining (Figure 3A). Production of collagen type I (COL1 ), collagen type II (COL2) and aggrecan (ACAN) was assessed after 7 days of micromass culture by qRT-PCR (Figure 4A). A half media change was performed every 3-4 days, where only standard recombinant growth factor was replenished but no extra PODS® were added.

Alcian blue staining after 21 days of micromass culture revealed that there was increased GAG production with standard rBMP-2 and rBMP-7 as well as with pBMP- 2 and pBMP-7 compared with growth media only or with pEmpty (Figure 3A). No significant differences in staining were detected between recombinant vs PODS®- treatment, or between BMP-2 and BMP-7.

Generally, COL1A1 , COL2A1 and ACAN mRNA expression was up-regulated in chondrocytes cultured with either standard recombinant or PODS® formulations of BMP-2 and BMP-7, when normalising to cells cultured in growth media only (Figure 4A). COL1 A1 mRNA expression was up-regulated by a similar amount with standard recombinant and pBMP-2 (9-fold and 10-fold, respectively). However, COL1A1 was more strongly up-regulated by standard rBMP-7 compared with pBMP-7 (110-fold and 17-fold, respectively). Conversely, up-regulation of ACAN mRNA was greater with pBMP-2 or pBMP-7 (8-fold and 5-fold, respectively), compared with standard rBMP2 and rBMP-7 (both 3-fold). Lastly, there was a 14-fold and 280-fold up-regulation in COL2A1 mRNA expression with standard rBMP-2 and rBMP-7, respectively, compared with a 4-fold up-regulation with pBMP-2 and no changes observed with pBMP-7. There was little change in COL1 , COL2 or ACAN mRNA expression with pEmpty relative cells cultured in growth media only. Changes in Alcian blue staining and mRNA were also measured in chondrocytes cultured with a dose range of pBMP-2 or pBMP-7 (Figure 3B, Figure 4B and Figure 4C, respectively).

A half media change was performed every 3-4 days with no extra PODS® crystals added. Alcian blue staining was observed at a range of PODS® crystals (25-100 ng/ml equivalent), with no trend observed between staining intensity and amounts of crystals (Figure 3B). There was a trend towards a dose-dependent increase in mRNA expression relative to 25 ng/ml equivalent of PODS® treatment (Figure 4B and Figure 4C). For COL1 A1 , there was approximately a 2-fold and 4-fold increase with 50 ng/ml and 75 ng/ml of pBMP-2 and pBMP-7, respectively. With 100 ng/ml, there was a 3- fold increase with pBMP-2, and no change with pBMP-7.

ACAN mRNA expression increased by 1 .5-2-fold with 50 ng/ml and 75 ng/ml of pBMP- 2, relative to 25 ng/ml pBMP-2, with no change for 100 ng/ml of pBMP-2. There was no change in ACAN mRNA expression with any dose of pBMP-7. Lastly, COL2A1 mRNA increased between 1.5-3-fold, with 50, 75 and 100 ng/ml pBMP-2 treatment, and 8.5-fold and 3.5-fold with 50 or 75 ng/ml of pBMP-7, respectively.

Example 11 - PODS® BMP-2 and PODS® BMP-7 promote in vivo chondral defect healing

C57BL/6 mice were subjected to a full-thickness cartilage defect, after which 6.75x105 PODS® crystals (18.75 ng equivalent) in PBS or 0.5% hydrogel were administered by intra-articular injection. Mice were sacrificed four and eight weeks post-surgery, and joints were sectioned and stained with Safranin O (Figure 5). Sections were quantitatively assessed for evidence of inflammation and the extent of cartilage repair and osteoarthritis in the joints using the Modified Pineda score and Makin score, respectively. At four weeks post-surgery there was no unambiguous trend and no significant difference in the damage repair between the different groups with either scoring system (Figures 6A and 6B), with the mean score fluctuating between 8.25 and 11.2 (Pineda score) and 9.8 and 12 (Mankin Score).

At eight weeks, there was a statistically significant reduction in the Modified Pineda score and the Mankin score in mice receiving pBMP-2 in 0.5% hydrogel or pBMP-7 in 0.5% hydrogel compared with the pooled four week data, with mean scores fluctuating between 4.0 and 9.0 (Pineda score) and 7.2 and 11 .36 (Mankin score) (Figures 6C and 6D). This indicates that there was a significant improvement in joint repair, and a significant reduction in osteoarthritis with both PODS® treatment groups, therefore demonstrating a biological effect.

OA is a degenerative disorder which currently lacks an effective early intervention to repair cartilage defects and halt progression of OA. With important roles in cartilage homeostasis and repair, BMP-2 and BMP-7 are promising disease-modifying candidates to treat OA and have previously been shown to be chondroprotective. The present invention demonstrates the efficacy and safety of pBMP-2 and pBMP-7 in vitro as well as in vivo to promote cartilage repair.

The in vitro experiments demonstrate that pBMP-2 and pBMP-7 promote chondrogenesis, ECM synthesis and proliferation, with results that are similar to standard recombinant growth factors. It is not possible to directly compare these results, due to innate differences between these culture systems. Whilst addition of standard recombinant growth factor provides an instant source of functionally active protein, PODS® must first be degraded before incorporated growth factor can be slowly released into the media. Proteases must be synthesized by cells to trigger release from PODS®, meaning that there is a lag phase before released growth factor accumulates to activate a biological response. In some of our experiments, a media change was performed every 3 days which would reduce the growth factor released from PODS® crystals by half in the culture system. Therefore, it is particularly striking that PODS® and standard recombinant growth factor have produced comparable results in these experiments.

Culturing primary chondrocytes with pBMP-2 or pBMP-7 up-regulated ECM marker genes (collagen and aggrecan) and synthesis of GAGs, which play crucial roles in the function of articular cartilage. Chondrogenic differentiation through Alcian blue staining with pBMP-2 supports previously published data. Whilst Alcian blue staining did not reveal any differences between PODS® growth factor and standard recombinant growth factor, there were some changes in mRNA expression. Expression of ACAN was higher with both pBMP-2 and pBMP-7 compared with standard recombinant growth factor, whereas expression of COL2A1 was higher for standard rBMP-2 and rBMP-7 compared with pBMP-2 and pBMP-7. Induction of COL1A1 was similar for both pBMP-2 and rBMP-2, whereas for BMP-7, standard recombinant protein generated a much higher increase in expression compared with PODS®-incorporated protein. This is likely to be due to the replenishment of standard recombinant on Day 3 during the half media change, with no replacement of PODS®. The half media change for the PODS®-containing wells would have removed half of the released growth factor from the culture system, which would take time to replace compared to the instant replenishment for standard recombinant growth factor-containing wells. In the future, this experiment may provide a better comparison between treatment groups (and better predict in vivo applications) by performing the PODS® treatment without a media change and not replenishing standard recombinant growth factor.

The effect of not changing the media during the culture period on PODS®-containing wells is illustrated by the real-time proliferation results. BMP-2 and BMP-7, respectively, were continuously released from PODS® crystals enabling sustained cellular proliferation over a period of two weeks, after an initial lag phase of four days. Cartilage metabolism is relatively slow in comparison with other tissues (17), therefore this lag phase is likely to be because degradation of PODS® is triggered by secreted proteases, which must be synthesized and secreted by cells before growth factor is released to trigger proliferation.

The proliferative activity of PODS® was in stark contrast to standard recombinant BMP-2 and BMP-7 counterparts, which only promoted chondrocyte proliferation for the first two days of culture. Here, growth factors were not replenished during the experiment, and due to their short half-life and fragility were quickly degraded, leading to loss of biological activity. Furthermore, the dose-dependent effect of PODS® growth factor in this system was evident, with the highest doses of PODS® leading to the most proliferation. This emphasizes the advantages of PODS® in this culture system without a media change, as PODS® constantly release functional growth factor at physiologically relevant levels over a sustained period of time. pBMP-2 and pBMP-7 crystals, respectively, were injected into the intra-articular knee joint space in mice. Analysis of the joints 8 weeks after administering treatment demonstrated that both pBMP-2 and pBMP-7 improved healing of the chondral defect and reduced signs of OA (as assessed through the Modified Pineda and Mankin scoring systems), with no adverse side effects observed. This suggests that pBMP-2 and pBMP-7 are efficacious and safe to administer in either PBS or 0.5% hydrogel formulations.

Intra-articular injection of just BMP-7 directly to the knee has been previously investigated as a treatment for OA in Phase I and Phase II clinical trials, but has proved to be unsuccessful (NCT00456157 - Intra-articular injection of OP-1 to affected knee (1 .0 ml) using ultrasound or fluoroscopy guidance in an outpatient setting (dose escalation study), NCT01111045 - Single intra-articular knee injection of OP-1 , and NCT01133613 - single intra-articular knee injection of OP-1). The Phase I trial of BMP-7 showed a trend towards a symptom response for knee OA with a lack of dose- limiting toxicity (18). However, the results from the Phase II studies were not published and no further studies have been announced. BMP-7 was also initially approved by the FDA under the Humanitarian Device Exemption programme as OP- 1 Putty (or Osigraft), a product for bone fusion during spinal surgery. However, the product was later withdrawn.

Currently there are no BMP-2 products in clinical trials for OA. However, BMP-2 applied to an ACS carrier is approved by the Food and Drug Administration as Infuse™ Bone Graft, and by the European Medicines Agency as InductOs™. This product is indicated for certain spinal fusion procedures, sinus augmentations, alveolar ridge augmentations, and for treating acute, stabilised open tibial shaft fractures. However, its approval has not been without controversy, with adverse side effects and complications reported such as excessive and/or off-target ossification, which may be due to the high dosage required (12 mg BMP-2 in a 1 .5 mg/ml solution) (19,20).

The present invention provides a convenient, off-the-shelf outpatient therapy for cartilage defects and early OA, addressing a currently unmet medical need and reducing the healthcare burden. Furthermore, the sustained release effect of the formulation according to the present invention provides therapeutic efficacy at much lower doses, preventing adverse side effects and improving cost-effectiveness.

Administering pBMP-2 and/or pBMP-7 compositions to repair cartilage defects according to the present invention had been demonstrated to be efficacious and suitable for the prevention and treatment of articular cartilage disorders. It is well within the knowledge and skills of a person skilled in the art to make any modifications to the methods and examples of the invention which are non-limiting and provided herein by way of illustration only.

REFERENCES

1. Hunter DJ, McDougall JJ, Keefe FJ. The symptoms of OA and the genesis of pain. Rheum Dis Clin North Am 2008;34:623-643.

2. De Luca F, Barnes KM, Uyeda JA, De-Levi S, Abad V, Palese T et al. Regulation of growth plate chondrogenesis by bone morphogenetic protein-2. Endocrinology 2001 ;142:430-436.

3. Chubinskaya S, Hurtig M, Rueger DC. OP-1/BMP-7 in cartilage repair. Int Orthop 2007; 31:773- 781.

4. Takahashi T, Muneta T, Tsuji K, Sekiya I. BMP-7 inhibits cartilage degeneration through suppression of inflammation in rat zymosan-induced arthritis. Cell Tissue Res 2011 ;344:321-332.

5. Badlani N, Oshima Y, Healey R, Coutts R, Amiel D. Use of Bone Morphogenic Protein-7 as a Treatment for Osteoarthritis. Clin Orthop 2009;467:3221-3229.

6. Blaney Davidson EN, Vitters EL, van Lent PLEM, van de Loo FAJ, van den Berg WB, van der Kraan PM. Elevated extracellular matrix production and degradation upon bone morphogenetic protein-2 (BMP-2) stimulation point toward a role for BMP-2 in cartilage repair and remodeling. Arthritis Res Ther 2007;9:R102.

7. Li X, Yi W, Jin A, Duan Y, Min S. Effects of sequentially released BMP-2 and BMP-7 from PELA microcapsule-based scaffolds on the bone regeneration. Am J Transl Res 2015;7:1417-1428.

8. Wegman F, Bijenhof A, Schuijff L, Oner FC, Dhert WJA, Alblas J. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo. Eur Cell Mater 2011 ;21:230- 242; discussion 242.

9. Mori H, Ito R, Nakazawa H, Sumida M, Matsubara F, Minobe Y. Expression of Bombyx mori cytoplasmic polyhedrosis virus polyhedrin in insect cells by using a baculovirus expression vector, and its assembly into polyhedra. J Gen Virol 1993;74 ( Pt 1 ):99— 102.

10. Ikeda K, Nakazawa H, Shimo-Oka A, Ishio K, Miyata S, Hosokawa Y et al. Immobilization of diverse foreign proteins in viral polyhedra and potential application for protein microarrays. Proteomics 2006;6:54-66. Nishishita N, Ijiri H, Takenaka C, Kobayashi K, Goto K, Kotani E et al. The use of leukemia inhibitory factor immobilized on virus-derived polyhedra to support the proliferation of mouse embryonic and induced pluripotent stem cells. Biomaterials 2011 ;32:3555-3563. Matsumoto G, Ueda T, Sugita Y, Kubo K, Mizoguchi M, Kotani E et al. Polyhedral microcrystals encapsulating bone morphogenetic protein 2 improve healing in the alveolar ridge. J Biomater Appl 2015 ;30 : 193-200. Eltawil NM, De Bari C, Achan P, Pitzalis C, Dell’accio F. A novel in vivo murine model of cartilage regeneration. Age and strain-dependent outcome after joint surface injury. Osteoarthritis Cartilage 2009;17:695-704. Pineda S, Pollack A, Stevenson S, Goldberg V, Caplan A. A Semiquantitative Scale or Histologic Grading of Articular Cartilage Repair. Cells Tissues Organs 1992;143:335-340. Mankin HJ, Dorfman H, Lippiello L, Zarins A. Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data. J Bone Joint Surg Am 1971 ;53:523-537. Ostergaard K, Petersen J, Andersen CB, Bendtzen K, Salter DM. Histologic/histochemical grading system for osteoarthritic articular cartilage: reproducibility and validity. Arthritis Rheum 1997;40:1766-1771. Hayes DW, Brower RL, John KJ. Articular cartilage. Anatomy, injury, and repair. Clin Podiatr Med Surg 2001 ;18:35-53. Hunter DJ, Pike MC, Jonas BL, Kissin E, Krop J, McAlindon T. Phase 1 safety and tolerability study of BMP-7 in symptomatic knee osteoarthritis. BMC Musculoskelet Disord 2010;11:232. Hustedt JW, Blizzard DJ. The Controversy Surrounding Bone Morphogenetic Proteins in the Spine: A Review of Current Research. Yale J Biol Med 2014;87:549-561. Skovrlj B, Marquez-Lara A, Guzman JZ, Qureshi SA. A review of the current published spinal literature regarding bone morphogenetic protein-2: an insight into potential bias. Curr Rev Musculoskelet Med 2014;7:182-188.

Claims

CLAIMS:

1 . A composition comprising a polyhedrin protein and at least one growth factor.

2. A composition according to claim 1 wherein the polyhedrin protein is a complex and forms a crystal scaffold.

3. A composition according to claim 1 or 2 wherein the growth factor is selected from one or more of OP-2, OP-3, BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP- 9, BMP-10, BMP-11 , BMP-12, BMP-13, BMP-15, BMP-16, BMP-17. BMP-18, DPP, Vgl, Vgr, 60A protein, GDF-1 , GDF-2, GDF- 3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9,GDF-10, GDF-11 , GDF-12, CDMP-1 , CDMP-2, CDMP-3,

NODAL, UNIVIN, SCREW, ADMP, NEURAL, and amino acid sequence variants thereof.

4. A composition according to any preceding claim wherein the growth factor is BMP-2.

5. A composition according to any one of claims 1 to 3 wherein the growth factor is BMP-7.

6. A composition according to any one of claims 1 to 3 wherein the composition comprises BMP-7 and BMP-2.

7. A composition according to any preceding claim for use in the treatment of musculoskeletal conditions, disorders, diseases or injury in a subject.

8. A composition for use according to claim 7 wherein the musculoskeletal condition is an injury, disease or disorder of the cartilage, meniscus or ACL.

9. A composition for use according to claim 7 wherein the disease is osteoarthritis or intervertebral disc degeneration.

10. A composition for use according to any one of claims 7 to 9 wherein treatment is due to promoting sustained cellular proliferation and/or production of extracellular matrix at the site of administration.

11 . A composition for use according to any one of claims 7 to 9 wherein treatment is due to one or more of increased production of ECM components such as GAGs, hyaluronic acid, fibronectin, or chondroitin sulphate, and, upregulation in the expression of ECM genes such as COLIA1 , COL2A1 and ACAN mRNA in chondrocytes, promoting chondrogenesis, ECM synthesis, cell proliferation and healing of chondral defects at the site of administration.

12. A composition for use according to any one of claims 7 to 11 wherein the subject is a mammal.

13. A composition for use according to claim 12 wherein the mammal is selected from humans, horses, dogs, cats and livestock.

14. A pharmaceutical formulation comprising the composition according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier selected from a physiological solution comprising any one or a combination of glucose, dextrose, normal saline, phosphate buffered saline (PBS) or Ringer's solution.

15. A composition according to any one of claims 1 to 6 or a pharmaceutical formulation according to claim 14 formulated in a gel, hydrogel, tablet, capsule, liquid, injectable solution, suspension or powder.

16. A delivery system comprising the composition according to any one of claims 1 to 6 or the pharmaceutical formulation according to claim 14 wherein the growth factor is released from the polyhedrin at the site of administration in a sustained manner, in a delayed manner or in a gradient-like manner.

17. A delivery system according to claim 16 wherein the delivery is at the site of musculoskeletal injury, osteoarthritis, intervertebral disc degeneration, meniscal tears, or injury of the ACL.

18. A method comprising the use of the composition according to any one of claims 1 to 6 in the prevention and/or treatment of cartilage or meniscal defects, conditions, disorders, diseases or injury, or injury of the ACL.

19. A method according to claim 18 wherein the disease is osteoarthritis or intervertebral disc degeneration.

20. A method according to claim 18 or 19 wherein the composition is administered by intra-articular injection or surgical intervention into the cartilage or into the synovial fluid surrounding the cartilage.

21. A method of inducing chondrocyte proliferation and ECM production in vivo to produce or accelerate cartilage formation, regenerate cartilage, or prevent cartilage degradation, in the treatment of cartilage injury or disease, comprising the use of the composition according to any one of claims 1 to 6 or a pharmaceutical formulation according to claim 14.