Elvire Gouze

Gene delivery to cartilage defects using coagulated bone marrow aspirate

The long-term goal of the present study is to develop a clinically applicable approach to enhance natural repair mechanisms within cartilage lesions by targeting bone marrow-derived cells for genetic modification. To determine if bone marrow-derived cells infiltrating osteochondral defects could be transduced in situ, we implanted collagen–glycosaminoglycan (CG) matrices preloaded with adenoviral vectors containing various marker genes into lesions surgically generated in rabbit femoral condyles. Analysis of the recovered implants showed transgenic expression up to 21 days; however, a considerable portion was found in the synovial lining, indicating leakage of the vector and/or transduced cells from the matrix. As an alternative medium for gene delivery, we investigated the feasibility of using coagulated bone marrow aspirates. Mixture of an adenoviral suspension with the fluid phase of freshly aspirated bone marrow resulted in uniform dispersion of the vector throughout, and levels of transgenic expression in direct proportion to the density of nucleated cells in the ensuing clot. Furthermore, cultures of mesenchymal progenitor cells, previously transduced ex vivo with recombinant adenovirus, were readily incorporated into the coagulate when mixed with fresh aspirate. These vector-seeded and cell-seeded bone marrow clots were found to maintain their structural integrity following extensive culture and maintained transgenic expression in this manner for several weeks. When used in place of the CG matrix as a gene delivery vehicle in vivo, genetically modified bone marrow clots were able to generate similarly high levels of transgenic expression in osteochondral defects with better containment of the vector within the defect. Our results suggest that coagulates formed from aspirated bone marrow may be useful as a means of gene delivery to cartilage and perhaps other musculoskeletal tissues. Cells within the fluid can be readily modified with an adenoviral vector, and the matrix formed from the clot is completely natural, native to the host and is the fundamental platform on which healing and repair of mesenchymal tissues is based.

Lentiviral-mediated gene delivery to synovium: potent intra-articular expression with amplification by inflammation

Clinical translation of gene-based therapies for arthritis could be accelerated by vectors capable of efficient intra-articular gene delivery and long-term transgene expression. Previously, we have shown that lentiviral vectors transduce rat synovium efficiently in vivo. Here, we evaluated the functional capacity of transgene expression provided by lentiviral-mediated gene delivery to the joint. To do this, we measured the ability of a lentiviral vector containing the cDNA for human interleukin-1 receptor antagonist (LV-hIL-1Ra) to suppress intra-articular responses to IL-1beta. Groups of rats were injected in one knee with 5 x 10(7) infectious units of LV-IL-1Ra. After 24 h, a range of doses of fibroblasts (3 x 10(3), 10(4), 3 x 10(4), or 10(5) cells) genetically modified to overexpress IL-1beta was injected into both knees. Intra-articular delivery of LV-hIL-1Ra strongly prevented swelling in all treated knees, even in those receiving the greatest dose of IL-1beta(+) cells. Cellular infiltration, cartilage erosion, and invasiveness of inflamed synovium were effectively prevented in LV-hIL-1Ra-treated knees and were significantly inhibited in contralateral joints. Beneficial effects were also observed systemically in the lentivirus-treated animals. Interestingly, intra-articular expression of the IL-1Ra transgene was found to increase in relation to the number of IL-1beta(+) cells injected. Further experiments using GFP suggest this is due to the proliferation of cells, stably modified by the integrative lentivirus, in response to inflammatory stimulation.

Exogenous glucosamine globally protects chondrocytes from the arthritogenic effects of IL-1β

The effects of exogenous glucosamine on the biology of articular chondrocytes were determined by examining global transcription patterns under normal culture conditions and following challenge with IL-1β. Chondrocytes isolated from the cartilage of rats were cultured in several flasks either alone or in the presence of 20 mM glucosamine. Six hours later, one-half of the cultures of each group were challenged with 10 ng/ml IL-1β. Fourteen hours after this challenge, RNA was extracted from each culture individually and used to probe microarray chips corresponding to the entire rat genome. Glucosamine alone had no observable stimulatory effect on the transcription of primary cartilage matrix genes, such as aggrecan, collagen type II, or genes involved in glycosaminoglycan synthesis; however, glucosamine proved to be a potent, broad-spectrum inhibitor of IL-1β. Of the 2,813 genes whose transcription was altered by IL-1β stimulation (P < 0.0001), glucosamine significantly blocked the response in 2,055 (~73%). Glucosamine fully protected the chondrocytes from IL-1-induced expression of inflammatory cytokines, chemokines, and growth factors as well as proteins involved in prostaglandin E2 and nitric oxide synthesis. It also blocked the IL-1-induced expression of matrix-specific proteases such as MMP-3, MMP-9, MMP-10, MMP-12, and ADAMTS-1. The concentrations of IL-1 and glucosamine used in these assays were supraphysiological and were not representative of the arthritic joint following oral consumption of glucosamine. They suggest, however, that the potential benefit of glucosamine in osteoarthritis is not related to cartilage matrix biosynthesis, but is more probably related to its ability to globally inhibit the deleterious effects of IL-1β signaling. These results suggest that glucosamine, if administered effectively, may indeed have anti-arthritic properties, but primarily as an anti-inflammatory agent.

Intra‐articular gene delivery and expression of interleukin‐1Ra mediated by self‐complementary adeno‐associated virus

The adeno‐associated virus (AAV) has many safety features that favor its use in the treatment of arthritic conditions; however, the conventional, single‐stranded vector is inefficient for gene delivery to fibroblastic cells that primarily populate articular tissues. This has been attributed to the inability of these cells to convert the vector to a double‐stranded form. To overcome this, we evaluated double‐stranded self‐complementary (sc) AAV as a vehicle for intra‐articular gene delivery.

Postnatal Soluble FGFR3 Therapy Rescues Achondroplasia Symptoms and Restores Bone Growth in Mice

A recombinant soluble fibroblast growth factor receptor 3 (FGFR3) restored normal skeletal growth and prevented disease-related complications in a mouse model of achondroplasia. Receptor Decoy Restores Bone Growth Achondroplasia is a rare disease where bone growth is stunted and cartilage does not form correctly. It is caused by a mutation in the gene that encodes fibroblast growth factor receptor 3 (FGFR3), which leads to overactive receptor signaling. Yet, despite knowing the cause, a treatment has not been discovered. In an innovative approach, Garcia and colleagues used receptor “decoys” to prevent the FGF ligand from binding its mutated receptor, thus interrupting the signaling cascade and restoring bone growth in mice. Normal mice or mice with the mutation in the gene encoding FGFR (Fgfr3ach/+) were treated with recombinant, soluble FGFR3 (sFGFR3) or a vehicle control for 3 weeks. More than 60% of Fgfr3ach/+ mice that were left untreated died during the treatment period, whereas only 12% of sFGFR3-treated mice died from achondroplasia-related complications, such as respiratory distress or paraplegia. In the surviving Fgfr3ach/+ animals, those treated with sFGFR3 had normal body and tail lengths, normal rib cage development, and decreased spinal and skull deformities—all similar to their healthy, wild-type counterparts. Untreated transgenic animals suffered from the defects common to achondroplasia: shortened stature, abnormal rib cage structure, and spinal compression, leading to paraplegia and bladder dysfunction. The sFGFR3 therapy was not toxic to the animals and did not affect reproduction (in fact, by increasing pelvis size in treated transgenic females, litter sizes were normal). Additional preclinical studies will be needed to see if this is a viable long-term treatment for achondroplasia, but with a long half-life and promising early outcomes in animals, this FGFR decoy may be a viable postnatal treatment for translation. Achondroplasia is a rare genetic disease characterized by abnormal bone development, resulting in short stature. It is caused by a single point mutation in the gene coding for fibroblast growth factor receptor 3 (FGFR3), which leads to prolonged activation upon ligand binding. To prevent excessive intracellular signaling and rescue the symptoms of achondroplasia, we have developed a recombinant protein therapeutic approach using a soluble form of human FGFR3 (sFGFR3), which acts as a decoy receptor and prevents FGF from binding to mutant FGFR3. sFGFR3 was injected subcutaneously to newborn Fgfr3ach/+ mice—the mouse model of achondroplasia—twice per week throughout the growth period during 3 weeks. Effective maturation of growth plate chondrocytes was restored in bones of treated mice, with a dose-dependent enhancement of skeletal growth in Fgfr3ach/+ mice. This resulted in normal stature and a significant decrease in mortality and associated complications, without any evidence of toxicity. These results describe a new approach for restoring bone growth and suggest that sFGFR3 could be a potential therapy for children with achondroplasia and related disorders.

scAAV-Mediated Gene Transfer of Interleukin 1-Receptor Antagonist to Synovium and Articular Cartilage in Large Mammalian Joints

With the long-term goal of developing a gene-based treatment for osteoarthritis (OA), we performed studies to evaluate the equine joint as a model for adeno-associated virus (AAV)-mediated gene transfer to large, weight-bearing human joints. A self-complementary AAV2 vector containing the coding regions for human interleukin-1-receptor antagonist (hIL-1Ra) or green fluorescent protein was packaged in AAV capsid serotypes 1, 2, 5, 8 and 9. Following infection of human and equine synovial fibroblasts in culture, we found that both were only receptive to transduction with AAV1, 2 and 5. For these serotypes, however, transgene expression from the equine cells was consistently at least 10-fold higher. Analyses of AAV surface receptor molecules and intracellular trafficking of vector genomes implicate enhanced viral uptake by the equine cells. Following delivery of 1 × 1011 vector genomes of serotypes 2, 5 and 8 into the forelimb joints of the horse, all three enabled hIL-1Ra expression at biologically relevant levels and effectively transduced the same cell types, primarily synovial fibroblasts and, to a lesser degree, chondrocytes in articular cartilage. These results provide optimism that AAV vectors can be effectively adapted for gene delivery to large human joints affected by OA.

Gene delivery of TGF-β1 induces arthrofibrosis and chondrometaplasia of synovium in vivo.

To understand the cellular and molecular events contributing to arthrofibrosis, we used an adenovirus to deliver and overexpress transforming growth factor-beta 1 (TGF-β1) cDNA (Ad.TGF-β1) in the knee joints of immunocompromised rats. Following delivery, animals were killed periodically, and joint tissues were examined macroscopically and histologically. PCR-array was used to assay alterations in expression patterns of extracellular matrix (ECM)-associated genes. By days 5 and 10, TGF-β1 induced an increase in knee diameter and complete encasement of joints in dense scar-like tissue, locking joints at 90° of flexion. Histologically, massive proliferation of synovial fibroblasts was seen, followed by their differentiation into myofibroblasts. The fibrotic tissue displaced the normal architecture of the joint capsule and fused with articular cartilage. RNA expression profiles showed high levels of transcription of numerous MMPs, matricellular and ECM proteins. By day 30, the phenotype of the fibrotic tissue had undergone chondrometaplasia, indicated by cellular morphology, matrix composition and >100-fold increases in expression of collagen type II and cartilage link protein. Pre-labeling of articular cells by injection with recombinant lentivirus containing eGFP cDNA showed fibrotic/cartilaginous tissues appeared to arise almost entirely from local proliferation and differentiation of resident fibroblasts. Altogether, these results indicate that TGF-β1 is a potent inducer of arthrofibrosis, and illustrate the proliferative potential and plasticity of articular fibroblasts. They suggest the mechanisms causing arthrofibrosis share many aspects with tumorigenesis.

Gait and behavior in an IL1β‐mediated model of rat knee arthritis and effects of an IL1 antagonist

Interleukin‐1 beta (IL1β) is a proinflammatory cytokine that mediates arthritic pathologies. Our objectives were to evaluate pain and limb dysfunction resulting from IL1β over‐expression in the rat knee and to investigate the ability of local IL1 receptor antagonist (IL1Ra) delivery to reverse‐associated pathology. IL1β over‐expression was induced in the right knees of 30 Wistar rats via intra‐articular injection of rat fibroblasts retrovirally infected with human IL1β cDNA. A subset of animals received a 30u2009µl intra‐articular injection of saline or human IL1Ra on day 1 after cell delivery (0.65u2009µg/µl hIL1Ra, nu2009=u20097 per group). Joint swelling, gait, and sensitivity were investigated over 1 week. On day 8, animals were sacrificed and joints were collected for histological evaluation. Joint inflammation and elevated levels of endogenous IL1β were observed in knees receiving IL1β‐infected fibroblasts. Asymmetric gaits favoring the affected limb and heightened mechanical sensitivity (allodynia) reflected a unilateral pathology. Histopathology revealed cartilage loss on the femoral groove and condyle of affected joints. Intra‐articular IL1Ra injection failed to restore gait and sensitivity to preoperative levels and did not reduce cartilage degeneration observed in histopathology. Joint swelling and degeneration subsequent to IL1β over‐expression is associated limb hypersensitivity and gait compensation. Intra‐articular IL1Ra delivery did not result in marked improvement for this model; this may be driven by rapid clearance of administered IL1Ra from the joint space. These results motivate work to further investigate the behavioral consequences of monoarticular arthritis and sustained release drug delivery strategies for the joint space.

In vitro gene transfer to chondrocytes and synovial fibroblasts by adenoviral vectors.

The major requirement of a successful gene transfer is the efficient delivery of an exogenous therapeutic gene to the appropriate cell type with subsequent high or regulated levels of expression. In this context, viral systems are more efficient than nonviral systems, giving higher levels of gene expression for longer periods. For the application of osteoarthritis (OA), gene products triggering anti-inflammatory or chondroprotective effects are of obvious therapeutic utility. Thus, their cognate genes are candidates for use in the gene therapy of OA. In this chapter, we describe the preparation, the use, and the effect of the transduction of chondrocytes or synovial fibroblasts with an adenoviral vector encoding the cDNA for glutamine: fructose-6-phosphate amidotransferase (GFAT). This is intended to serve as an example of a technology that can be used to evaluate the biological effects of overexpression of other cDNAs.

Gene therapy for rheumatoid arthritis

Rheumatoid arthritis (RA) is a disabling, painful disorder affecting 1% of the world’s population. Although the aetiology of RA remains unknown, recent advances in understanding its pathophysiology have led to the characterisation of several proteins whose activities may be anti-arthritic. Clinical application of such proteins has greatly improved the treatment of RA, but the disease remains incurable and difficult to manage in a substantial number of patients. Thus, there are continued efforts to develop new therapeutic strategies. Because RA is a chronic condition, effective treatment will probably require the presence of therapeutic agents for extended periods of time. In the case of proteins, this is problematic. Gene therapy may offer a solution to this problem. Experimental studies have confirmed the feasibility, efficacy and safety of gene therapy for the treatment of animal models of arthritis. Several different approaches have shown promise in this regard, including gene transfer to the synovial lining cells of individual joints and the systemic delivery of genes to extra-articular locations. One unexpected finding has been the ‘contralateral effect’ in which gene delivery to one joint of an animal with polyarticular disease leads to improvement of multiple joints. Investigation of this phenomenon has led to interest in cell trafficking and the genetic modification of antigen-presenting cells (APC). The first Phase I clinical trial tested the feasibility and safety of ex vivo gene transfer to the synovial lining of human joints. This clinical trial has been successfully completed and two other Phase I trials are in progress. A Phase II study is now being planned to investigate the efficacy of gene transfer to the joints of patients with early stage RA.