Osteoarthritis (OA) is a chronic degenerative disease of the joints of vertebrates. Pain is the predominant presenting symptom of OA. Vertebrates with OA, including humans, mammalian animals, particularly athletic-horses, dogs and cats, have pain that typically worsens with weight bearing and activity and improves with rest, as well as morning stiffness and gelling of the involved joints after periods of inactivity. Such signs and symptoms of the disease often culminate in reductions in quality of life. The disease is more frequent in a subset of the joints of the body, particularly the hips, knees, shoulders, elbows, intercarpus and intertarsus joints.
Diarthrodial joints are organs of locomotion of vertebrates. They achieve almost frictionless motion, thanks to the unique biological, chemical and mechanical properties of the articular cartilage that covers the articulating surfaces of the long bones. Articulation occurs within a synovial cavity, or joint space, whose intimal surface is lined by synovium and within which there normally exits a small volume of lubricating synovial fluid. Certain joints, such as the knee have additional, nonarticular cartilaginous structures, known as menisci. The synovium lines a fibrous capsule, beyond which lies the musculature and ligamentous extra-articular supporting structures of the joint.
Current OA impact in humans is tremendous and rivals that of ischemic heart disease in many regards. As baby boomers reach late adulthood and the obesity epidemic rages on, OA will assume an even greater impact on society. In younger populations, athletes participating in contact sports or activities, such as excessive running, are also at high risk for the development of OA.
In veterinary medicine, OA is very common in horses and dogs but is also recognised in cats, albeit at a lower extend.
In the equine industry, lameness due to joint injury and disease is the most prevalent cause of diminished athletic function and wastage in racing horses. Together, joint injury and joint disease represent a large majority of the equine clinician's caseload. Equine OA can originate from various causes, with trauma and concomitant synovitis being the most common causes of lack of performance in horses. Often OA stems from overuse or conformational inadequacies that predispose an athletic horse to inappropriate biomechanical forces on cartilage.
In the dog, OA is one of the most common chronic musculoskeletal diseases and causes of lameness. It is frequently secondary to congenital or acquired musculoskeletal disorders. Several arthropathies can affect the young dog and lead to secondary OA, including joint dysplasia, osteochondrosis dissecans, un-united anconeal process and patellar luxation. In addition to developmental abnormalities, there are many acquired musculoskeletal disorders associated to cartilage deterioration. In some cases, trauma may directly induce an isolated chondral lesion that can be the onset of an extended and progressive degenerative lesion. After ligamentous lesions, cartilage injuries may appear some weeks later as a consequence of joint instability. Intra-articular fractures are often complicated by secondary cartilage degradation, as a consequence of incomplete fracture reduction. Joint luxation or luxation reduction are commonly complicated by ligament and capsule damage and/or cartilage lesions. Obesity is more and more understood as an important risk factor for OA in dogs as it is in human medicine.
OA is characterized by biochemical and enzymatic changes, cartilage fragmentation and loss, osteophytes formation and bony sclerosis. Although the causes of OA are not completely understood, biochemical stresses affecting the articular cartilage and subchondral bone, biochemical changes in the articular cartilage and synovial membrane, and genetic factors are all important in OA pathogenesis.
Most often, the inflammatory process begins in the synovium, cartilage, joint capsule or subchondral bone and quickly initiates a cascade of inflammatory mediators from the primary tissue of insult. This often causes a “domino effect” of the inflammatory process into the secondary tissues that in turn release inflammatory mediators.
Regardless of the species, the molecular and cellular inflammatory events associated to OA involve the release of metabolites of arachidonic acid in the cell membrane. This in turn initiates pain by means of prostaglandins. Degradation of hyaluronic acid in the joint fluid results from chemoattractants and by-products of the inflammatory pathway, lysosomal enzymes, and non-lysomal enzymes elaborated by injured synoviocytes, and oxygen-derived free radicals from neutrophils and macrophages.
Degeneration of the articular cartilage is considered the sine qua non of OA. Gross findings include fibrillation, erosion and wear lines in the articular cartilage. Histological characteristics include superficial fibrillation, which can progress to form vertical clefts down to subchondral bone. Often the proteoglycan content of the articular cartilage is reduced along with the breakdown of collagen. This results in increased water uptake in cartilage leading to a biomechanically “softer” cartilage surface. These findings can also be accompanied by chondrocyte necrosis and eventual full-thickness loss of articular cartilage. Pathological changes may occur in associated structures as well. Subchondral bone sclerosis commonly accompanies cartilage degeneration, and the demarcation between hyaline and calcified articular cartilage becomes penetrated with blood vessels. Chronic progression of these changes leads to formation of periarticular lipping at the joint margins due to progressive overgrowth of cartilage and subchondral bone along the borders of articulations. The synovial lining and fibrous layer of the joint capsule are altered in OA. The synovium becomes congested, discoloured, and thickened. Histologically, synoviocytes appear hypertrophic, and lymphoplasmacytic cells and macrophages may be present in the subintimal stroma of the synovial tissues.
Numerous medical treatments have been used extensively in the treatment of OA. To date, most treatments have been directed towards lowering and then maintaining a decreased degree of inflammation within damaged joints. Relatively little attention has been focused on therapeutic agents that actually protect the joint tissues and which have been classified by the International League Against Rheumatism (ILAR) guidelines as “disease modifying” OA drugs (DMOADs). Therapy with this class of drugs should, in theory, prevent, retard or reverse morphologic cartilaginous lesions of OA.
Non steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids have been the primary mode of anti-inflammatory therapy. Although non steroidal anti-inflammatory drugs often provide symptomatic relief, little protection and regeneration is afforded to the articular cartilage, nor do the drugs modify the underlying disease process. Many NSAIDs are also associated to significant incidence of undesirable side effects, making their long-term use problematic. Corticosteroids also are commonly used to treat OA and are powerful mediators of reducing pain and inflammation. However, untoward effects on articular cartilage, including impaired chondrocyte activity, decreased glycosaminoglycans and proteoglycans content and decreased cartilage elasticity are reported.
In veterinary practice, aforementioned therapies are sometimes combined for an additive, if not a synergistic, response to joint injury. Often NSAIDs or steroids are combined with hyaluronic acid treatments to improve the viscoelasticity of the joint fluid and boundary lubrication of the intra-articular soft tissues. Patients are also frequently placed on parenteral as well as oral polysulphated glycosaminoglycans (PSGAGs) to sustain or promote chondrocyte metabolic activity and inhibit the detrimental effects of cytokines or prostaglandins on cartilage. While no well-controlled clinical studies have evaluated this “shotgun” approach, each drug has beneficial effects and no adverse effects have been reported to date from using these many different modes of therapy.
Therapeutic intervention in OA is further hindered, in part, by the inability to target therapeutic agents directly into the joints. Traditional oral, intravenous and intramuscular routes are relatively ineffective, because small molecules enter the joint space by passive diffusion and large molecules (such as proteins) are excluded from the joint space. Although intra-articular administration bypasses these limitations, the half-life of most agents directly administered into the joint space remains short and frequent intra-articular injections are needed to sustain biologic activities for prolonged treatments of chronic diseases.
In conclusion, as to date, no therapeutic agent has effectively, and without side effects, eliminated the progression of OA. There is therefore a crying medical need for truly innovative DMOADs strategies for both human and veterinary applications.
Recently, Osteogenic Protein 1 (OP-1), alias Bone Morphogenetic Protein 7 (BMP-7), previously known to promote bone formation and healing, has been demonstrated to play a significant role in articular cartilage regeneration and repair, potentially acting as a DMOADs. Various studies have shown that, when exposed to BMP-7, mesenchymal cells have the potential to differentiate into cells that behave phenotypically as chondrocytes. These cells, both in vitro and in vivo, produce matrix with type II collagen and proteoglycans specific for articular cartilage. This finding has been confirmed in various animal models including a canine large full-thickness osteochondral defect model. The repair tissue obtained has been observed to have hyaline-like appearance and to be maintained over long-term animal studies.
However, the use of BMP-7 for OA therapy is not without limitations and potential complications. Recombinant purified BMP-7 protein in solution is not providing the expected therapeutical benefit when merely injected into a joint because of rapid elimination from the joint space through the synovial vascularisation. Short half-life of the protein within the articulation requires the recombinant BMP-7 protein to be included into an appropriate biological carrier material to ensure slow release and sustained local concentration. The biological carrier further secures the implant stability and physical network for cellular and vascular colonisation leading to cartilage formation. The high concentrations of recombinant protein to be included in the biological carrier (e.g., 340 μg of OP-1 implant administered twice by intra-articular injection one week apart in a sheep post traumatic experimental OA model (Hurtig M B et al., Proceedings Combined Orthopaedic Research Society, 070, October 2004), 350 μg of BMP-7 in bovine-derived type-I collagen device to repair an articular cartilage defect in a canidae surgically induced full thickness osteochondral defect (Cook S. D. et al., J. Bone Joint Surgery, 2003, 85(3): 116-123)). In practical terms, the cost and the complexity of complexing large amounts of purified protein with an appropriate biological carrier makes the procedure non economically viable in veterinary medicine. Further, from a safety standpoint, the use of large quantities of a powerful osteoinductive protein raises the possibility of ectopic bone formation, particularly if the site of implantation is not well contained.
Gene transfer is an attractive strategy to circumvent limitations facing purified recombinant proteins. As gene can be delivered and expressed locally within joints, highest concentrations of therapeutical proteins can be produced in situ within the joint, thereby reducing the likelihood of unwanted side effects to a minimal as non-target organs will receive less exposure. The synovium is an attractive target tissue for gene expression because of its large surface and its direct contact with the joint space. Although the articular cartilage is another available target tissue within the joint, lack of vector penetration through the extracellular matrix is considered an important technical limitation.
Two methods of transferring genes to joints can be considered: ex vivo and in vivo gene transfers.
Ex vivo gene transfer refers to the harvest of cells, their in vitro genetic modification and their subsequent back grafting into the joint. Indeed, accelerated cartilage repair has been demonstrated following transplantation of chondrocytes transduced ex vivo with an Ad5 expressing BMP-7 in an equine cartilage defect model. As this technology allows genetic manipulations outside of the body it is associated to an increased safety profile. However, the approach is facing multiple practical issues as it is time and resources consuming for harvesting, manipulating and re-implanting cells. Further, the longevity of the recombinant protein expression in the chondrocytes transplants remains a limiting factor and requires either very high initial doses or repeat treatments. Clinical applicability of such a strategy is complex and cumbersome, dramatically limiting commercialization potential in veterinary medicine.
In contrast, in vivo gene transfer refers to the direct targeting of synoviocytes or chondrocytes in the joint itself, which is achievable at the clinical level using appropriate vectors. However, this later strategy is also restricted by significant limitations, the exact nature of which depends on the specific vector system that is considered.
Adenoviruses possess many advantages as gene therapy vectors, including the ease to generate high titers of recombinant viruses, the wide range of cell types, including non dividing cells, that are susceptible to efficient transduction by such viruses. The so-called first generation recombinant human adenovirus serotype 5 (Ad5) is based on the deletion of the E1 and potentially the E3 regions of the viral genome. These modifications provide loci for transgene insertion but also result in the prevention of late genes activation, upon which viral replication in target species depends. Such advantages have led to the widespread application of Ad5 vectors both in preclinical and clinical studies. However, the death of a human patient exposed to very large Ad5 doses in 1999 has tarnished the safety record of adenovirus vectors. As of today, the safety of Ad5 vectors administered systemically is considered questionable by many.
However, significant limitations or uncertainties face the utility of this vector for clinical use in joints. The trigger of an anti Ad5 immune response may interfere with the transgene delivery and long-term expression but also may cause pathology, usually inflammatory. Immune and inflammatory interferences have indeed been noticed with recombinant Ad5 delivered intra-articulary in mice, rats, rabbits or horses. Although batch to batch variations and purity may be involved, the amount of viral particles injected intra-articularly appears a key aspect driving Ad5 associated inflammation for intra-articular gene therapy. As the efficacy of the Ad5 vector to trigger expression of the transgene within the joint can be proportional to the injected dose, the selection of the appropriate balance between efficacy and safety for articular gene therapy remains a technical challenge.
The definition of an Ad5 dosage compatible with long-term expression of the recombinant protein within the joint is not clear from existing literature. Indeed, when an Ad5 expressing IGF1 was injected into an equine fetlock joint, significant expression of IGF1 was detectable in the synovial fluid only for those horses receiving very high doses (50×1010 viral particles (VP)) (Goodrich L R et al., Gene Therapy, 2006, 13: 1253-1262). No significant expression was noticed with lower doses. This high Ad5 dosage is associated to an increase in white blood cells (WBC) in the synovial fluid, demonstrating a significant local inflammation. Under these conditions, expression of IGF1 peaked at day 4 post injection and declined thereafter. Temporal declines in transgene expression over a period of 2-4 weeks have been observed in other studies using Ad5 as a delivery vector. Although the transient elevation could positively impact cartilage healing in the first 4 weeks and possibly longer, the need for vectors that express proteins for months rather than weeks is considered preferable by many authors in the literature. Indeed, most investigators seeking prolonged gene expression are currently re-orienting their efforts to alternative existing vector systems, such as adeno-associated viruses (AAVs) or lentiviruses.
Prior reports of in vivo usage of recombinant BMP-7 protein for cartilage repair indicated that very high amounts of recombinant protein were required (e.g., 340 μg of OP-1 implant administered twice by intra-articular injection one week apart in a sheep post traumatic experimental OA model (Hurtig M B et al., Proceedings Combined Orthopaedic Research Society, 070, October 2004), 350 μg of BMP-7 in bovine-derived type-I collagen device to repair an articular cartilage defect in a canidae surgically induced full thickness osteochondral defect (Cook S. D. et al., J. Bone Joint Surgery, 2003, 85(3): 116-123)). These amounts are significantly higher than the peak amounts (73 ng/ml) reported in studies using the aforementioned Ad5 IGF1 (Goodrich L R et al., Gene Therapy, 2006, 13: 1253-1262). As a consequence, prior experience with Ad5 vectors for joint gene therapy do not support the use of an Ad5 BMP-7 vector to reach therapeutical concentrations in vivo.
Of further relevance, prior use of high doses of Ad5 BMP7 for in vivo bone formation in immunocompetent rats was unsuccessful whereas the exact same recombinant virus was able to trigger significant bone formation in immunocompromized rats (Li J Z et al., Gene Therapy, 2003, 10: 1735-1743). This example further illustrates the complexity of the prediction of efficacy of an Ad5 BMP7 in vivo.
The technical problem to be addressed is to ensure articular cartilage repair and thereby slow down and potentially reverse osteoarthritis disease evolution in mammals.
Articular cartilage covers the articulating surfaces of the portions of bones in joints. The cartilage allows movement in joints without direct bone-to-bone contact, thereby preventing wearing down and damage of opposing bone surfaces. Articular cartilage has no tendency to ossification. The cartilage surface appears smooth and pearly macroscopically, and is finely granular under high power magnification. Such cartilage is referred to as hyaline cartilage, as opposed to fibrocartilage and elastic cartilage. Articular cartilage appears to derive its nutriment partly from the vessels of the neighboring synovial membrane, partly from those of the bone that it covers. Articular cartilage is associated with the presence of Type II and Type IX collagen and various well-characterized proteoglycans, and with the absence of Type X collagen, which is associated with endochondral bone formation. For a detailed description of articular cartilage micro-structure, see, for example, Aydelotte and Kuettner, Conn. Tiss. Res. 18:205 (1988); Zanetti et al., J. Cell Biol. 101:53 (1985); and Poole et al., J. Anat. 138:13 (1984).
Other types of permanent cartilage in adult mammals include fibrocartilage and elastic cartilage. In fibrocartilage, the mucopolysaccharide network is interlaced with prominent collagen bundles and the chondrocytes are more widely scattered than in hyaline cartilage. Interarticular fibrocartilages are found in those joints which are most exposed to violent concussion and subject to frequent movement, e.g., the meniscus of the knee. Examples of such joints include the temporo-mandibular, sterno-clavicular, acromio-clavicular joints. Elastic cartilage contains collagen fibers that are histologically similar to elastin fibers. Such cartilage is found in the human body in the auricle of the external ear, the Eustachian tubes, the cornicula laryngis, and the epiglottis.
Bone Morphogenetic Protein-7 (BMP-7, or Osteogenic Protein-1, OP-1) is a member of the Transforming Growth Factor-β (TGF-β) superfamily. BMP-7 binds to activin receptors types I and II, but not to TGF-β receptors type I, II and III. Monomeric BMP-7 has a molecular weight of 17 to 19 kDa and was identified by its ability to induce ectopic bone formation. BMP-7 polypeptide is secreted as a homodimer with an apparent molecular weight of approximately 35-36 kDa.
However, because BMP-7 has a short half live in vivo (approximately 30 min), maintenance of a sustained level of exogenous protein in the circulation following injection of the purified protein requires multiple short-interval administrations, creating a very significant practical challenge. The cost of such a multi-injection therapy is too high to be applicable in veterinary medicine. Although gene delivery has been successfully promoted as an alternative to protein therapy for various diseases treatment, it's applicability for osteoarthritis prevention and/or treatment through BMP-7 polypeptide expression in vivo has not been proposed previously, and its potential effectiveness remains uncertain. Indeed, the low molecular weight of the BMP-7 homodimer (i.e., approximately 35 kDa) would theoretically allow for rapid glomerular filtration. Whether or not levels of BMP-7 expressed in vivo could reach therapeutically effective plasma concentrations cannot be predicted or determined from the existing literature. To further complicate the evaluation of in vivo-expressed BMP proteins, results can be variable depending on the immune status of the treated animal, with significant differences between immune competent and incompetent animals. Thus, when considered collectively as a whole, the literature does not teach whether levels of BMP-7 expressed in vivo could reach plasma concentrations that would be therapeutically useful.
Citation or identification of any document in this application does not constitute an admission that such document is available as prior art to the present invention.
USPTO Backgrounds
USPTO Backgrounds