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 × 10(11) 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.

A simple, lanthanide-based method to enhance the transduction efficiency of adenovirus vectors.

Based upon the powerful bridging and charge-masking properties of lanthanide cations (Ln3+), we have investigated their use to improve the transduction efficiency of adenovirus vectors. Using a luciferase marker gene, it was possible to increase transgene expression by the murine mesenchymal stem cell line C3H10T(1/2) by up to four log orders when using very low multiplicities of infection in conjunction with Ln3+; La3+ was superior to Gd3+, Y3+ and Lu3+ in this regard. All Ln3+ were more effective than Ca2+. Flow cytometry, using a green fluorescent protein marker gene, confirmed that La3+ increased both the percentage of transduced cells and the level of transgene expression per cell. Transduction of primary cultures of a variety of different mesenchymal cells from human, rabbit, bovine and rat sources, as well as gene transfer to synovium and muscle in vivo, was also greatly enhanced. Our findings suggest that this lanthanide-based method holds much promise for expediting both experimental and clinical applications of gene transfer with adenoviral vectors.

cDNA sequencing and expression analysis of Dicentrarchus labrax heme oxygenase-1.

The liver cDNA encoding heme oxygenase--1 (HO-1) was sequenced from European sea bass (Dicentrarchus labrax) (accession number no. EF139130). The HO-1 cDNA was 1250 bp in nucleotide length and the open reading frame encoded 277 amino acid residues. The deduced amino acid sequence of the European sea bass had 75% and 50% identity with the amino acid sequences of tetraodontiformes (Tetraodon nigroviridis and Takifugu rubripes) and human HO-1 proteins, respectively. A short hydrophobic transmembrane domain at the C--terminal region was found, and four histidine residues were highly conserved, including human his25 that is essential for HO catalytic activity. RT-PCR of mRNA from eight different European sea bass tissues revealed that, in a homeostatis state, the heme oxygenase--1 was abundant in the spleen and liver but not in the brain.

Gene therapeutic approaches-transfer in vivo.

Osteoarthritis (OA) is common, debilitating, expensive, incurable and very difficult to treat. Gene transfer to the synovial linings of affected joints is a promising strategy for achieving sustained, therapeutic, intraarticular concentrations of anti-arthritic gene products. This is not reasonably possible with existing, alternative technologies. The present review summarizes progress in achieving direct, in vivo intraarticular gene delivery and expression. Numerous non-viral vectors have been evaluated for their ability to transfect the synovia of experimental animals following intraarticular injection. None have given more than low levels of temporary transgene expression and many are inflammatory. Several viral vectors, however, are very effective in this regard and successfully treat experimental models of OA. Adeno-associated virus has been used in a phase I study for the gene therapy of rheumatoid arthritis. Its use in a clinical trial for treating OA is pending.

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.

Gene therapy for rheumatoid arthritis.

Advances in understanding the biology of rheumatoid arthritis (RA) have opened new therapeutic avenues. One of these, gene therapy, involves the delivery to patients of genes encoding anti-arthritic proteins. This approach has shown efficacy in animal models of RA, and the first human, phase I trial has just been successfully completed. Hand surgery featured prominently in this pioneering study, as a potentially anti-arthritic gene encoding the interleukin-1 receptor antagonist was transferred to the metacarpophalangeal joints of subjects with RA one week before total joint arthroplasty. This study has confirmed that it is possible to transfer genes safely to human joints. It should pave the way for additional application of gene therapy to arthritis and other orthopaedic conditions.

Cloning of Wap65 in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) and expression in sea bass tissues.

The complementary DNA encoding WAP65 protein was cloned from the liver of two fish species sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). Full-length cDNA sequences were obtained from reverse transcribed total RNA, followed by 5' and 3' rapid amplification of cDNA end (RACE) experiments. The full-length cDNA sequence of D. labrax is 1709bp and the coding sequence is flanked by a 67bp 5'-UTR and a 358bp 3'-UTR. The full-length cDNA sequence of S. aurata is 1599bp, and the coding sequence is flanked by a 48bp 5'-UTR and a 273bp 3'-UTR. The deduced amino acid putative primary sequences are composed of 427 and 425 amino acid residues for D. labrax and S. aurata, respectively. They display high homologies with previously described fish WAP65 and other hemopexin-like proteins from rabbit (Oryctolagus cuniculus). Expression of Wap65 has proved to be a natural physiological adaptive answer of teleost fish to warm temperature acclimation. In all fish species studied to date, Wap65 was found expressed mainly by the liver, although other tissues seem able to express Wap65 in response to a warm temperature acclimation, in a specie specific manner. Here, we investigate the tissue specific expression of Wap65 in D. labrax and S. aurata in response to a warm temperature acclimation, by RT-PCR analysis.

Gene therapy for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a painful chronic disorder. Conventional therapies are palliative, not curative. Advances in the understanding of the pathophysiology of RA have led to the development of new therapeutic strategies, including gene therapy. Multiple studies in several different animal models provide proof supporting the use of gene therapy in arthritis. A phase I clinical trial has already been performed successfully on nine women with end-stage RA in the United States, and two other trials are in progress. Limited duration of gene expression impedes the development of a clinically useful genetic treatment for arthritis.

Osteoarthritis gene therapy.

Osteoarthritis (OA) is the Western world's leading cause of disability. It is incurable, costly and responds poorly to treatment. This review discusses strategies for treating OA by gene therapy. As OA affects a limited number of weight-bearing joints and has no major extra-articular manifestations, it is well suited to local, intra-articular gene therapy. Possible intra-articular sites of gene transfer include the synovium and the cartilage. Most experimental progress has been made with gene transfer to synovium, a tissue amenable to genetic modification by a variety of vectors, using both in vivo and ex vivo protocols. The focus so far has been upon the transfer of genes whose products enhance synthesis of the cartilaginous matrix, or inhibit its breakdown, although there is certainly room for alternative targets. It is possible to build a convincing case implicating interleukin-1 (IL-1) as a key mediator of cartilage loss in OA, and the therapeutic effects of IL-1 receptor anatagonist (IL-1Ra) gene transfer have been confirmed in three different experimental models of OA. As transfer of IL-1Ra cDNA to human arthritic joints has already been accomplished safely, we argue that clinical studies of intra-articular IL-1Ra gene transfer in OA are indicated and should be funded. Of the available vector systems, recombinant adeno-associated virus may provide the best combination of safety with in vivo delivery using current technology.

Adenovirus-mediated gene transfer of glutamine: fructose-6-phosphate amidotransferase antagonizes the effects of interleukin-1beta on rat chondrocytes.

OBJECTIVE: To determine whether overexpression of glutamine: fructose-6-phosphate amidotransferase (GFAT) in synoviocytes will antagonize the response to interleukin-1beta (IL-1beta) of chondrocytes and synovial fibroblasts in co-culture. METHODS: Synovial fibroblasts from the rat were transduced by an adenovirus carrying the cDNA for GFAT and then co-cultured with rat chondrocytes encapsulated in alginate beads. Following challenge with 1, 5, or 10 ng/ml of IL-1beta for 24 h, proteoglycan synthesis by the chondrocytes was determined by incorporation of Na2(35)SO4. Production of nitric oxide (NO) and prostaglandin E2 (PGE2) were monitored by assay of conditioned medium from the co-culture. RESULTS: IL-1beta treatment of untransduced-synoviocyte/chondrocyte co-cultures resulted in markedly decreased proteoglycan synthesis by the chondrocytes, and increased NO and PGE2 levels in the culture medium. In contrast, adenovirus-mediated transfer of GFAT in synoviocytes prevented both the decrease in chondrocyte proteoglycan synthesis and increases in NO and PGE2 provoked by IL-1beta. CONCLUSIONS: Our study suggests that in a synoviocyte/chondrocyte co-culture system, overexpression of GFAT by synoviocytes significantly inhibits subsequent stimulation by IL-1beta in vitro. Since GFAT is the rate limiting enzyme in the synthesis of intracellular glucosamine and its derivatives, these results may open new possibilities for osteoarthritis treatment.

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.