Gene transfer strategies for medical management of osteoarthritis have attracted the attention of scientists to the complex pathology of this chronic disease. Unlike other pharmacological treatments, gene therapy targets the disease process rather than the symptoms. 
Passing from parents to children, genes are building blocks of inheritance. They contain instructions for making proteins . If genes do not produce the right proteins in a correct way, a child can have a genetic disorder. Gene therapy is a molecular method aiming to replace defective or absent genes, or to counteract the undergoing overexpression. For this purpose, three techniques can be used: gene isolation, manipulations, and transferring to target cells. The most common form of gene therapy involves inserting a normal gene to replace an abnormal gene. Other approaches including repairing an abnormal gene and altering the degree to which a gene is turned on or off. Two basic methodologies are used to transfer vectors to target tissues; Ex vivo gene transfer and In-vivo gene transfer. One type of gene therapy in which the gene transfer takes place outside the patient’s body is called ex vivo gene therapy. This method of gene therapy is more complicated but it is possible to culture, test, and control the modified cells.
Significance and causes of osteoarthritis
Osteoarthritis (OA) is a degenerative joint disease which is the western world’s leading cause of pain and disability.   It is characterized by the progressive loss of normal structure and function of articular cartilage , the smooth tissue covering the end of the moving bones.  This chronic disease not only affects the articular cartilage but the subchondral bone, the synovium and periarticular tissues are other candidates.  People with OA can experience severe pain and limited motion. OA is mostly the result of natural aging of the joint due to biochemical changes in the extracellular matrix cartilage .  
Osteoarthritis is caused by mechanical factors such as obesity, joint trauma , mechanical overloading of joints or joint-instability.  Genetics is also a leading factor that contributes to OA. Studies have shown that they are the source of at least 50% of OA cases in the hands and hips . Since the degeneration of cartilage is an irreversible phenomenon, it is incurable, costly, and responds poorly to treatment.  Due to the prevalence of this disease, the repair and regeneration of articular cartilage has become a dominant area of research.  The growing number of patients suffering from osteoarthritis and the effectiveness of the treatment of these diseases has a great deal of attention to genetic-based therapeutic methods to cure and prevent progression of this chronic disease.
Vectors for osteoarthritis gene delivery
Various vectors have been developed to carry the therapeutic genes to cells. There are two broad categories of gene delivery vectors: Viral vectors , Involving viruses and nonviral agents, Such As polymers and liposomes . 
Viral vectors are more successful in their transfecting cells than in their life cycles. A virus infects the human by inserting its gene directly into its cells. This can be deadly, but the brilliant idea is to take advantage of this natural ability. The idea is to remove all the dangerous genes in the virus and inject the healthy human genes. So, viruses are inserting positive elements into the host cells while attacking them. 
While viral vectors are more effective in transferring genes, they are not fully appreciated in vivo because of their further adverse effects. Primarily, viral vectors induce an inflammatory response, which can cause minor side effects such as mild edema or serious ones like multisystem organ failure. It is also difficult to administer gene therapy repeatedly to the immune system. Furthermore, viruses may spread to other organs after intraarticular injection and this will be a significant disadvantage. However, the majority of these problems are associated with viral vectors. In Osteoarthritis gene therapy, ex vivo method makes it possible to transfect not only the cells of the synovial lining of joints but also articular chondrocytes and chondroprogenitor cells in cartilage. 
Non-viral methods involve complexing therapeutic DNA to various macromolecules including cationic lipids and liposomes , polymers , polyamines and polyethylenimines , and nanoparticles . FuGene 6  and modified cationic liposomes  are two non-viral gene delivery methods that have been used for cartilage delivery. FuGene 6 is a non-liposomal lipid formulation, which has been proven to be successful in transfecting a variety of cell lines. Liposomes have been shown to be an appropriate candidate for gene delivery, Where cationic liposomes are made to facilitate interaction with cell membranes and nucleic acids.  Viral vectors, non-viral ones, the risk of acquiring replication competence. They have the capacity to deliver a large amount of therapeutic genes repeatedly, and it is possible to produce them on a large scale. The most important of all, they do not elicit immune responses in the host organism. In Spite of HAVING advantages, non-viral vectors have-nots yet REPLACED viral vectors due to Relatively low efficiency and short-term transgene expression. 
Novel non-viral vectors for osteoarthritis gene delivery include polymeric vectors are still under investigation.
Target cells in osteoarthritis gene therapy
Codocyte in the OA therapy are autologous chondrocytes , Chondroprogenitor cells, Cells dans le synovial cavity,  and cells of adjacent tissues Such As muscle , tendons , ligaments , and meniscus . Development of cartilage function and structure can be achieved by:
- Inhibiting inflammatory and catabolic pathways
- Stimulating anabolic pathways to rebuild the matrix
- Impeding cell senescence
- Avoiding the pathological formation of osteophytes
- Prevention of apoptosis , and / or influencing several of these processes 
Approaches influençant Several of These processes Simultaneously-have aussi shown to be successful, like Transferring the combination of inhibitors of catabolism pathways and activators of anabolic events ( IGF-I / IL-1RA ), as well as That of activators of anabolic and proliferative processes (FGF-2 / SOX9 or FGF-2 / IGF-I). 
Gene defects leading to osteoarthritis
Osteoarthritis has a great degree of heritability.  Forms of osteoarthritis caused by single gene mutation have better chance of treatment by gene therapy.  Epidemiological studies have shown that a genetic component may be an important risk factor in OA.  Insulin-like growth factor I (IGF-1) genes, Transforming growth factor β, oligomeric matrix protein cartilage , bone morphogenetic protein , and other anabolic gene candidates are among the candidate genes for OA.  Genetic changes in OA can lead to defects of a structural protein such as collagenor changes in the metabolism of bone and cartilage. Rarely OA is regarded as a single disorder Following Mendelian inheritance being white Predominantly a multifactorial disease.
However, in the field of OA gene therapy, researches has more focused on gene transfer, rather than counteracting genetic abnormalities or polymorphisms . Genes, which help to protect and restore the matrix of articular cartilage, are attracting the most attention. These Genes are listed in Table 1 . Among all the candidates listed below, the proteins that block the actions of interleukin-1 (IL-1) or which promote the synthesis of cartilage matrix molecules have received the most experimental scrutiny. 
|Cytokine / cytokine antagonist||IL-1Ra, IL-1R, sTNFR, IL-4|
|Cartilage growth factor||IGF-1, FGF, BMPs, TGF, CGDF|
|Matrix breakdown inhibitor||TIMPs, PAIs, serpins|
|Signaling molecule / transcription factor||Smad, Sox-9, IkB|
|Extra cellular matrix molecule||Type II collagen, COMP|
|Free radical antagonist||Super Oxide Dismutase|
Interleukin-1 as a target in osteoarthritis
Researches suggest that among all potential mediators, a protein called interleukin-1 is by far the most potent cause of pain, joint inflammation and loss of cartilage associated with osteoarthritis.  A therapeutic gene used to treat the arthritic joins produces a second protein, which naturally counteracts the effect of interleukin-1.  The Interleukin 1 receptor antagonist (IL-1Ra), the natural agonist of IL-1, is a protein that binds non-productively to the cell surface of interleukin-1 receptor, therefore blocks the activities of IL-1 by preventing it is sending a signal to IL-1 receptor.  There are three main researches that prove the benefits of local IL-1Ra gene therapy in animal models of osteoarthritis . Series of experiments on canines, rabbits, and horses demonstrates that IL-1Ra gene therapy is safe and effective in animal models of OA, according to which recombinant human IL-1Ra strongly protects the articular cartilage from degenerative changes.   
Strategies for osteoarthritis gene therapy
In the context of OA, the most attractive intra-articular sites for the transfer of synovium and articular cartilage. The present invention relates to an intra-articular tissue, such as the synovium, which can be modified by a variety of vectors, using both in vivo and ex vivo protocols.
Gene transfer to synovium
The main purpose of this invention is to provide an endogenous source of therapeutic molecules ( Table-1 ) which can be used to diffuse the metabolism of adjacent tissues such as cartilage. Genes can be delivered to synovium in animal models of RA and OA by direct, in vivo injection of vector or by indirect, ex vivo methods involving autologous synovial cells, skin fibroblasts , or other cell types such as mesenchymal stem cells . The direct in vivo approach is intra-articular insertion of a vector to affect synovicytes. Vectors play crucial role in success of this method. The effect of different vectors for in vivo is summarized in table 2 :
|Non-viral Vectors||Short-term and less efficient transfection; many inflammatory|
|Retrovirus||No transduction of normal synovium; modest transduction of inflamed synovium|
|lentivirus||Extremely high transduction and transgene expression; no obvious side effects|
|adenovirus||High transduction efficiency; dose-dependent inflammatory response|
|Adeno-associated virus||Moderate levels of transduction of normal and inflamed synovium|
|Herpes simplex virus||Highly efficient transduction; cytotoxic|
The indirect ex vivo approach involves harvesting synovium, isolation and culture of synoviocytes, in vitro transduction, and injection of engineered synovitis in the joint. 
Gene transfer to cartilage
Contrary to the synoviocytes which are dividing cells and can be transduced in vivo using either liposomes or viral vectors, in vivo delivery of genes to chondrocytes is hindered by the dense extra cellular matrix that surrounds these cells. Chondrocytes are non-dividing cells, embedded in a network of collagens and proteoglycans ; These studies suggest that genes can be transferred to chondrocytes within normal cartilage by intraarticular injection of liposomes containing virus (HVJ-liposomes)  and adeno-associated virus.  
Most efficient methods of cartilage transfer have involved ex vivo strategies using chondrocytes or chondroprogenitor cells. Chondrocytes are genetically enhanced by transferring complementary DNA encoding IL-1RA , IGF-1 , or matrix break down inhibitors mentioned in Table 1 . As discussed before, the transplanted cells could serve as an intra- articular source of therapeutic molecules. 
One important issue related to human gene therapy is safety, particularly for the gene therapy of non-fatal diseases such as OA. The main concern is the high immunogenicity of certain viral vectors. Retroviral vectors integrate into the chromosomes of the cells they infect. There will be a chance of integrating a tumor suppressor gene or an oncogene , leading to virulent transformation of the cell.  In General [ quote needed ]
- Vectors in gene therapy
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