Expert Opinion on Biological Therapy
ISSN: 1471-2598 (Print) 1744-7682 (Online) Journal homepage: http://www.tandfonline.com/loi/iebt20
Gene therapy for human osteoarthritis: principles and clinical translation Henning Madry & Magali Cucchiarini To cite this article: Henning Madry & Magali Cucchiarini (2016) Gene therapy for human osteoarthritis: principles and clinical translation, Expert Opinion on Biological Therapy, 16:3, 331-346, DOI: 10.1517/14712598.2016.1124084 To link to this article: http://dx.doi.org/10.1517/14712598.2016.1124084
Accepted author version posted online: 21 Nov 2015. Published online: 15 Dec 2015. Submit your article to this journal
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Date: 18 September 2017, At: 01:23
EXPERT OPINION ON BIOLOGICAL THERAPY, 2016 VOL. 16, NO. 3, 331–346 http://dx.doi.org/10.1517/14712598.2016.1124084
REVIEW
Gene therapy for human osteoarthritis: principles and clinical translation Henning Madry and Magali Cucchiarini
Downloaded by [Australian Catholic University] at 01:23 18 September 2017
Center of Experimental Orthopaedics, Saarland University, Homburg/Saar, Germany ABSTRACT
ARTICLE HISTORY
Introduction: Osteoarthritis (OA) is the most prevalent chronic joint disease. Its key feature is a progressive articular cartilage loss. Gene therapy for OA aims at delivering gene-based therapeutic agents to the osteoarthritic cartilage, resulting in a controlled, site-specific, long-term presence to rebuild the damaged cartilage. Areas covered: An overview is provided of the principles of gene therapy for OA based on a PubMed literature search. Gene transfer to normal and osteoarthritic cartilage in vitro and in animal models in vivo is reviewed. Results from recent clinical gene therapy trials for OA are discussed and placed into perspective. Expert opinion: Recombinant adeno-associated viral (rAAV) vectors enable to directly transfer candidate sequences in human articular chondrocytes in situ, providing a potent tool to modulate the structure of osteoarthritic cartilage. However, few preclinical animal studies in OA models have been performed thus far. Noteworthy, several gene therapy clinical trials have been carried out in patients with end-stage knee OA based on the intraarticular injection of human juvenile allogeneic chondrocytes overexpressing a cDNA encoding transforming growth factor-beta-1 via retroviral vectors. In a recent placebo-controlled randomized trial, clinical scores were improved compared with placebo. These translational results provide sufficient reason to proceed with further clinical testing of gene transfer protocols for the treatment of OA.
Received 5 October 2015 Accepted 20 November 2015 Published Online 15 December 2015
1. Introduction Osteoarthritis (OA) is the most prevalent chronic joint disease, with a rising incidence caused by the aging population worldwide, among other factors.[1] From a clinical standpoint, pain and the gradual loss of the joint function are its major features. From a structural point of view, the continuing loss of the articular cartilage is the key characteristic of OA, together with pathological changes of the subchondral bone and all other structures of a joint.[2–4] As this loss of articular cartilage occurs gradually, novel biological therapies might be of particular value for the early stages of the disease when cartilage erosion is just starting. However, current therapeutic treatments intending to target articular cartilage destruction either via oral administration of different components of the articular cartilage extracellular matrix (ECM) such as glucosamine or chondroitin sulfate or via intra-articular injections have had varied success and do not halt the progression of the disease.[5,6] Gene therapy for OA aims at delivering gene-based treatments into the joint space, resulting in a controlled, site-specific, long-term presence of a therapeutic agent to protect and rebuild the damaged articular cartilage.[7] The aim CONTACT Henning Madry [email protected] Building 37, D-66421 Homburg/Saar, Germany © 2015 Taylor & Francis
KEYWORDS
Articular cartilage; chondrocyte; clinical trials; gene therapy; human osteoarthritis; vectors
of this review is to provide an overview of the principles of gene therapy for OA. Gene transfer to normal and osteoarthritic cartilage in vitro and in animal models in vivo is specifically reviewed. Recent results from the recent clinical gene therapy trials for knee OA are discussed and placed into perspective.
2. Articular cartilage and osteoarthritis Hyaline articular cartilage is the gliding tissue that covers the end of bones that articulate in a diarthrodial joint.[8] Its competent function depends on the structural integrity of the cartilage.[9] Articular cartilage consists of chondrocytes embedded in an extensive network of ECM and water. Importantly, cartilage does not have nerves, blood vessels or a lymphatic system.[10] The chondrocytes—the sole cell type in cartilage—contribute only ~5% to the total cartilage volume. They are aligned in three major zones: the superficial tangential zone with ellipsoidal cells that are aligned parallel to the surface; the middle zone with randomly distributed spherical chondrocytes; and the deep zone containing cells arranged in columns aligned perpendicular to the surface. A zone of calcified
Center of Experimental Orthopaedics, Saarland University Medical Center, Kirrberger Str. 100,
332
H. MADRY AND M. CUCCHIARINI
Article highlights ● ● ● ● ● ●
Gene therapy is a promising therapeutic approach for structural modification of osteoarthritis (OA). Principles of OA gene therapy are examined. Gene transfer has been chiefly used to promote cartilage regeneration. Significant progress has been achieved in the safety and efficiency of viral and nonviral gene delivery methods. Encouraging preliminary results have been reported from recent clinical gene therapy trials in knee OA. The main challenge for the clinical applications of gene therapy remains ensuring safety while guaranteeing effectiveness.
Downloaded by [Australian Catholic University] at 01:23 18 September 2017
This box summarizes key points contained in the article.
cartilage follows, containing smaller cells. Separated by the cement line, the subchondral bone plate and subarticular spongiosa form the adjacent subchondral bone.[11] After skeletal maturity, chondrocytes rarely divide, but continue to synthesize and maintain the ECM. The major structural macromolecules are type-II collagen and other collagens, proteoglycans and noncollagenous proteins. The collagen fibers form a fibrillar meshwork that provides tensile strength. Proteoglycans and noncollagenous proteins are attached to this meshwork.[12] They provide cartilage with stiffness in compression and resilience, because proteoglycans confine large quantities of water which contributes up to 80% of the cartilage wet weight. In the heterogeneous disorder of OA,[13] not only the articular cartilage, but all other structures of a joint are affected.[14] These mainly include the subchondral bone [15] and synovial membrane, but also the menisci, capsular structures, tendons and ligaments, and muscles.[16] OA is a multifactorial disease with a genetic background.[2,17,18] In general, a dysbalance between anabolic and catabolic events is causing the onset of the disease.[2–4,19] Pathological loading is an important secondary factor,[20] caused, for instance, by axial malalignment,[21] loss of meniscal tissue,[22] prearthrotic deformities such as acetabular dysplasia [23] and repetitive stress injury.[24] Initially, a cartilage edema results in a minor volume increase, accompanied by chondrocyte hypertrophy, apoptosis and necrosis in the superficial zone.[14] Cartilage fibrillations in the superficial zone occur, and the loss of proteoglycans in the upper parts of cartilage reduces its water content and thus the elastic properties of cartilage. This early OA is the first phase of the joint destruction.[16] Matrix cracks start extending into the middle zone, and vertical clefts form. The collagen fiber structure is progressively damaged, and the cartilage eroded. This is accompanied by synovial inflammation, characterized by the release of catabolic mediators such as IL-1 and TNF-α which may further promote
cartilage degeneration.[25,26] This cartilage destruction ensues, until, in late OA, the cartilage is eroded down to the subchondral bone which may be exposed and deformed (Figure 1).[14]
3. Principles of gene therapy for osteoarthritis As a genetic background for human OA remains largely elusive, gene therapy for this disease does not aim at replacing or repairing an abnormal gene which might cause the disease. Rather, gene transfer technologies are used to either overexpress therapeutic factors such as growth or transcription factors or to suppress the expression of genes that support the OA joint destruction. In general, two major avenues are followed to transfer gene carriers (vectors) into the appropriate target tissue of the joint (Figure 2). In vivo gene transfer aims at directly introducing the gene of interest into the damaged tissue. Ex vivo gene transfer, in contrast, relies on the genetic modification of a target cell population outside the patient’s body and therefore involves, to some degree, the manipulation of the target cells. Because OA mainly affects the articular cartilage, this tissue is the major target for all these approaches. OA is a heterogeneous and progressive disease with different
Figure 1. Macroscopic view of knee osteoarthritis. Shown is the right distal femur of a 72-year-old man. Note the large, illdefined osteoarthritic cartilage lesion extending over the entire medial femoral condyle (MFC, right side of the picture), together with osteoarthritic changes of the femoral trochlea (TF) and the formation of an osteophyte (O) in the medial border of the femoral trochlea. LFC: Lateral femoral condyle; SM: Synovial membrane.
Downloaded by [Australian Catholic University] at 01:23 18 September 2017
EXPERT OPINION ON BIOLOGICAL THERAPY
333
Figure 2. Principles of gene therapy for osteoarthritis. Two major avenues are followed to overexpress a therapeutic gene in the joint and deliver its therapeutic factor into the appropriate target tissue. Left side: the direct application of a gene vector in vivo allows for an introduction of the gene of interest into the damaged tissue. Shown is the direct application of recombinant adeno-associated viral (rAAV) vectors, the only class of vectors available to date that permits a direct transduction of human (osteoarthritic) articular chondrocytes within their native extracellular matrix (inset: fluorescent illumination of osteoarthritic human articular cartilage, showing the expression of red fluorescent protein—RFP—in rAAVRFP transduced clusters of human osteoarthritic chondrocytes). Right side: ex vivo gene transfer, in contrast, relies on the genetic modification of a target cell population outside the patient’s body and therefore involves, to some degree, the manipulation of the target cells. In the case of the clinical gene therapy trials for osteoarthritis, human juvenile allogeneic chondrocytes were transduced with a retroviral vector carrying a cDNA encoding transforming growth factor-beta-1 (TGFβ1). Transduced, TGF-β1 overexpressing cells were then irradiated and combined with untransduced, non-irradiated human juvenile allogeneic chondrocytes from the same donor. This blend of cells was intra-articularly injected into the affected knee joint. Note the largely paracrine effect of the secreted TGF-β1 protein (red circles) on the osteoarthritic cartilage.
Downloaded by [Australian Catholic University] at 01:23 18 September 2017
334
H. MADRY AND M. CUCCHIARINI
phenotypes that continuously evolve, eventually leading to common clinical and structural manifestation. When considering gene therapy for the treatment of OA, this heterogeneity needs to be taken into account. For example, when early OA is developing based on the loss of meniscal tissue or instability, these responsible changes have to be addressed as well. Or, if OA is based on a recent traumatic event, gene therapeutic approaches also need to address the concomitant high levels of inflammatory mediators, e.g. by simultaneously targeting the synovial membrane. From a structural standpoint, the early phases of OA are most interesting to treat with gene therapy approaches, since the osteoarthritic destruction did not yet entirely erode the entire articular cartilage. These early stages could thus provide a very important window of opportunity for gene-based treatments toward and a regenerative treatment of OA. Such a stage-dependent approach for gene therapy for OA has already been outlined elsewhere.[27] Yet, genes may be also overexpressed in other tissues such as the synovial membrane and its secreted products might secondarily affect the articular cartilage. For ex vivo approaches, such cell populations chiefly include articular chondrocytes, progenitor cells, fibroblasts, but also bone marrow aspirates.
4. Gene delivery methods to articular cartilage The availability of various, enhanced gene transfer methods has allowed to effectively target the chondrocytes in vitro and in situ/in vivo when the cells are surrounded by their dense ECM. Such procedures are based on either nonviral compounds or virus-derived vehicles that use natural entry pathways in cells. A variety of gene delivery vectors have been tested to transfer therapeutic genes into target cells. They may be categorized in two classes: (1) viral vectors based on genetically manipulated viruses and (2) nonviral gene transfer systems among which are liposomes and polymers. Traditionally, the majority of viral vectors and/or nonviral vectors have been unable to directly transduce the articular chondrocytes embedded within their native ECM. In contrast, they have been powerful candidates for a variety of ex vivo gene transfer approaches. Recombinant adeno-associated viral (rAAV) vectors are the only class of vectors available to date that permits effective, direct in vivo gene transfer approaches into the articular cartilage as such constructs are capable of transducing articular chondrocytes within their native ECM environment.
4.1. Nonviral vectors Nonviral vectors allow to complex DNA with formulations that include plasmids, cationic liposomes, peptides, DNA–ligand complexes and gene-gun technique. Diverse commercially available systems may be applied to target the chondrocytes or other populations of cells relevant to OA such as a lipid-based reagent FuGENE® 6 for chondrocytes [28] and a polyamine formulation (GeneJammer®) for mesenchymal stem cells (MSCs).[29] Nonviral gene transfer approaches are commonly employed for the ex vivo modification of cells [30] as their efficacy (~35–40%) is lower than those achieved with viral vectors and in light of the lack of success to modify chondrocytes directly in the cartilaginous ECM.
4.2. Viral vectors Vectors based on adenovirus allow for high in vitro gene transfer efficiencies and expression levels both in chondrocytes (between 90% and close to 100%) [31– 33] and in MSCs (up to 80%),[33] but their use in vivo leads to host immune responses while transgene expression remains brief (1 – 2 weeks), an issue to treat a progressive disease like OA. Of further note, intra-articular injection of such vectors mostly targets the synovial cells, without proper access to the articular cartilage.[34] Retroviral vectors are attractive carriers as they have an ability to stably integrate in the host genome for persistence of the transgene delivered. A drawback of this class of vectors is the possibility to activate tumor genes and to provoke insertional mutagenesis events upon integration. While employed to modify other cells and tissues, retroviral vectors may not be best suited to target quiescent chondrocytes as these vehicles only transduce dividing cells, allowing for relatively low efficiencies (
ISSN: 1471-2598 (Print) 1744-7682 (Online) Journal homepage: http://www.tandfonline.com/loi/iebt20
Gene therapy for human osteoarthritis: principles and clinical translation Henning Madry & Magali Cucchiarini To cite this article: Henning Madry & Magali Cucchiarini (2016) Gene therapy for human osteoarthritis: principles and clinical translation, Expert Opinion on Biological Therapy, 16:3, 331-346, DOI: 10.1517/14712598.2016.1124084 To link to this article: http://dx.doi.org/10.1517/14712598.2016.1124084
Accepted author version posted online: 21 Nov 2015. Published online: 15 Dec 2015. Submit your article to this journal
Article views: 290
View related articles
View Crossmark data
Citing articles: 8 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iebt20 Download by: [Australian Catholic University]
Date: 18 September 2017, At: 01:23
EXPERT OPINION ON BIOLOGICAL THERAPY, 2016 VOL. 16, NO. 3, 331–346 http://dx.doi.org/10.1517/14712598.2016.1124084
REVIEW
Gene therapy for human osteoarthritis: principles and clinical translation Henning Madry and Magali Cucchiarini
Downloaded by [Australian Catholic University] at 01:23 18 September 2017
Center of Experimental Orthopaedics, Saarland University, Homburg/Saar, Germany ABSTRACT
ARTICLE HISTORY
Introduction: Osteoarthritis (OA) is the most prevalent chronic joint disease. Its key feature is a progressive articular cartilage loss. Gene therapy for OA aims at delivering gene-based therapeutic agents to the osteoarthritic cartilage, resulting in a controlled, site-specific, long-term presence to rebuild the damaged cartilage. Areas covered: An overview is provided of the principles of gene therapy for OA based on a PubMed literature search. Gene transfer to normal and osteoarthritic cartilage in vitro and in animal models in vivo is reviewed. Results from recent clinical gene therapy trials for OA are discussed and placed into perspective. Expert opinion: Recombinant adeno-associated viral (rAAV) vectors enable to directly transfer candidate sequences in human articular chondrocytes in situ, providing a potent tool to modulate the structure of osteoarthritic cartilage. However, few preclinical animal studies in OA models have been performed thus far. Noteworthy, several gene therapy clinical trials have been carried out in patients with end-stage knee OA based on the intraarticular injection of human juvenile allogeneic chondrocytes overexpressing a cDNA encoding transforming growth factor-beta-1 via retroviral vectors. In a recent placebo-controlled randomized trial, clinical scores were improved compared with placebo. These translational results provide sufficient reason to proceed with further clinical testing of gene transfer protocols for the treatment of OA.
Received 5 October 2015 Accepted 20 November 2015 Published Online 15 December 2015
1. Introduction Osteoarthritis (OA) is the most prevalent chronic joint disease, with a rising incidence caused by the aging population worldwide, among other factors.[1] From a clinical standpoint, pain and the gradual loss of the joint function are its major features. From a structural point of view, the continuing loss of the articular cartilage is the key characteristic of OA, together with pathological changes of the subchondral bone and all other structures of a joint.[2–4] As this loss of articular cartilage occurs gradually, novel biological therapies might be of particular value for the early stages of the disease when cartilage erosion is just starting. However, current therapeutic treatments intending to target articular cartilage destruction either via oral administration of different components of the articular cartilage extracellular matrix (ECM) such as glucosamine or chondroitin sulfate or via intra-articular injections have had varied success and do not halt the progression of the disease.[5,6] Gene therapy for OA aims at delivering gene-based treatments into the joint space, resulting in a controlled, site-specific, long-term presence of a therapeutic agent to protect and rebuild the damaged articular cartilage.[7] The aim CONTACT Henning Madry [email protected] Building 37, D-66421 Homburg/Saar, Germany © 2015 Taylor & Francis
KEYWORDS
Articular cartilage; chondrocyte; clinical trials; gene therapy; human osteoarthritis; vectors
of this review is to provide an overview of the principles of gene therapy for OA. Gene transfer to normal and osteoarthritic cartilage in vitro and in animal models in vivo is specifically reviewed. Recent results from the recent clinical gene therapy trials for knee OA are discussed and placed into perspective.
2. Articular cartilage and osteoarthritis Hyaline articular cartilage is the gliding tissue that covers the end of bones that articulate in a diarthrodial joint.[8] Its competent function depends on the structural integrity of the cartilage.[9] Articular cartilage consists of chondrocytes embedded in an extensive network of ECM and water. Importantly, cartilage does not have nerves, blood vessels or a lymphatic system.[10] The chondrocytes—the sole cell type in cartilage—contribute only ~5% to the total cartilage volume. They are aligned in three major zones: the superficial tangential zone with ellipsoidal cells that are aligned parallel to the surface; the middle zone with randomly distributed spherical chondrocytes; and the deep zone containing cells arranged in columns aligned perpendicular to the surface. A zone of calcified
Center of Experimental Orthopaedics, Saarland University Medical Center, Kirrberger Str. 100,
332
H. MADRY AND M. CUCCHIARINI
Article highlights ● ● ● ● ● ●
Gene therapy is a promising therapeutic approach for structural modification of osteoarthritis (OA). Principles of OA gene therapy are examined. Gene transfer has been chiefly used to promote cartilage regeneration. Significant progress has been achieved in the safety and efficiency of viral and nonviral gene delivery methods. Encouraging preliminary results have been reported from recent clinical gene therapy trials in knee OA. The main challenge for the clinical applications of gene therapy remains ensuring safety while guaranteeing effectiveness.
Downloaded by [Australian Catholic University] at 01:23 18 September 2017
This box summarizes key points contained in the article.
cartilage follows, containing smaller cells. Separated by the cement line, the subchondral bone plate and subarticular spongiosa form the adjacent subchondral bone.[11] After skeletal maturity, chondrocytes rarely divide, but continue to synthesize and maintain the ECM. The major structural macromolecules are type-II collagen and other collagens, proteoglycans and noncollagenous proteins. The collagen fibers form a fibrillar meshwork that provides tensile strength. Proteoglycans and noncollagenous proteins are attached to this meshwork.[12] They provide cartilage with stiffness in compression and resilience, because proteoglycans confine large quantities of water which contributes up to 80% of the cartilage wet weight. In the heterogeneous disorder of OA,[13] not only the articular cartilage, but all other structures of a joint are affected.[14] These mainly include the subchondral bone [15] and synovial membrane, but also the menisci, capsular structures, tendons and ligaments, and muscles.[16] OA is a multifactorial disease with a genetic background.[2,17,18] In general, a dysbalance between anabolic and catabolic events is causing the onset of the disease.[2–4,19] Pathological loading is an important secondary factor,[20] caused, for instance, by axial malalignment,[21] loss of meniscal tissue,[22] prearthrotic deformities such as acetabular dysplasia [23] and repetitive stress injury.[24] Initially, a cartilage edema results in a minor volume increase, accompanied by chondrocyte hypertrophy, apoptosis and necrosis in the superficial zone.[14] Cartilage fibrillations in the superficial zone occur, and the loss of proteoglycans in the upper parts of cartilage reduces its water content and thus the elastic properties of cartilage. This early OA is the first phase of the joint destruction.[16] Matrix cracks start extending into the middle zone, and vertical clefts form. The collagen fiber structure is progressively damaged, and the cartilage eroded. This is accompanied by synovial inflammation, characterized by the release of catabolic mediators such as IL-1 and TNF-α which may further promote
cartilage degeneration.[25,26] This cartilage destruction ensues, until, in late OA, the cartilage is eroded down to the subchondral bone which may be exposed and deformed (Figure 1).[14]
3. Principles of gene therapy for osteoarthritis As a genetic background for human OA remains largely elusive, gene therapy for this disease does not aim at replacing or repairing an abnormal gene which might cause the disease. Rather, gene transfer technologies are used to either overexpress therapeutic factors such as growth or transcription factors or to suppress the expression of genes that support the OA joint destruction. In general, two major avenues are followed to transfer gene carriers (vectors) into the appropriate target tissue of the joint (Figure 2). In vivo gene transfer aims at directly introducing the gene of interest into the damaged tissue. Ex vivo gene transfer, in contrast, relies on the genetic modification of a target cell population outside the patient’s body and therefore involves, to some degree, the manipulation of the target cells. Because OA mainly affects the articular cartilage, this tissue is the major target for all these approaches. OA is a heterogeneous and progressive disease with different
Figure 1. Macroscopic view of knee osteoarthritis. Shown is the right distal femur of a 72-year-old man. Note the large, illdefined osteoarthritic cartilage lesion extending over the entire medial femoral condyle (MFC, right side of the picture), together with osteoarthritic changes of the femoral trochlea (TF) and the formation of an osteophyte (O) in the medial border of the femoral trochlea. LFC: Lateral femoral condyle; SM: Synovial membrane.
Downloaded by [Australian Catholic University] at 01:23 18 September 2017
EXPERT OPINION ON BIOLOGICAL THERAPY
333
Figure 2. Principles of gene therapy for osteoarthritis. Two major avenues are followed to overexpress a therapeutic gene in the joint and deliver its therapeutic factor into the appropriate target tissue. Left side: the direct application of a gene vector in vivo allows for an introduction of the gene of interest into the damaged tissue. Shown is the direct application of recombinant adeno-associated viral (rAAV) vectors, the only class of vectors available to date that permits a direct transduction of human (osteoarthritic) articular chondrocytes within their native extracellular matrix (inset: fluorescent illumination of osteoarthritic human articular cartilage, showing the expression of red fluorescent protein—RFP—in rAAVRFP transduced clusters of human osteoarthritic chondrocytes). Right side: ex vivo gene transfer, in contrast, relies on the genetic modification of a target cell population outside the patient’s body and therefore involves, to some degree, the manipulation of the target cells. In the case of the clinical gene therapy trials for osteoarthritis, human juvenile allogeneic chondrocytes were transduced with a retroviral vector carrying a cDNA encoding transforming growth factor-beta-1 (TGFβ1). Transduced, TGF-β1 overexpressing cells were then irradiated and combined with untransduced, non-irradiated human juvenile allogeneic chondrocytes from the same donor. This blend of cells was intra-articularly injected into the affected knee joint. Note the largely paracrine effect of the secreted TGF-β1 protein (red circles) on the osteoarthritic cartilage.
Downloaded by [Australian Catholic University] at 01:23 18 September 2017
334
H. MADRY AND M. CUCCHIARINI
phenotypes that continuously evolve, eventually leading to common clinical and structural manifestation. When considering gene therapy for the treatment of OA, this heterogeneity needs to be taken into account. For example, when early OA is developing based on the loss of meniscal tissue or instability, these responsible changes have to be addressed as well. Or, if OA is based on a recent traumatic event, gene therapeutic approaches also need to address the concomitant high levels of inflammatory mediators, e.g. by simultaneously targeting the synovial membrane. From a structural standpoint, the early phases of OA are most interesting to treat with gene therapy approaches, since the osteoarthritic destruction did not yet entirely erode the entire articular cartilage. These early stages could thus provide a very important window of opportunity for gene-based treatments toward and a regenerative treatment of OA. Such a stage-dependent approach for gene therapy for OA has already been outlined elsewhere.[27] Yet, genes may be also overexpressed in other tissues such as the synovial membrane and its secreted products might secondarily affect the articular cartilage. For ex vivo approaches, such cell populations chiefly include articular chondrocytes, progenitor cells, fibroblasts, but also bone marrow aspirates.
4. Gene delivery methods to articular cartilage The availability of various, enhanced gene transfer methods has allowed to effectively target the chondrocytes in vitro and in situ/in vivo when the cells are surrounded by their dense ECM. Such procedures are based on either nonviral compounds or virus-derived vehicles that use natural entry pathways in cells. A variety of gene delivery vectors have been tested to transfer therapeutic genes into target cells. They may be categorized in two classes: (1) viral vectors based on genetically manipulated viruses and (2) nonviral gene transfer systems among which are liposomes and polymers. Traditionally, the majority of viral vectors and/or nonviral vectors have been unable to directly transduce the articular chondrocytes embedded within their native ECM. In contrast, they have been powerful candidates for a variety of ex vivo gene transfer approaches. Recombinant adeno-associated viral (rAAV) vectors are the only class of vectors available to date that permits effective, direct in vivo gene transfer approaches into the articular cartilage as such constructs are capable of transducing articular chondrocytes within their native ECM environment.
4.1. Nonviral vectors Nonviral vectors allow to complex DNA with formulations that include plasmids, cationic liposomes, peptides, DNA–ligand complexes and gene-gun technique. Diverse commercially available systems may be applied to target the chondrocytes or other populations of cells relevant to OA such as a lipid-based reagent FuGENE® 6 for chondrocytes [28] and a polyamine formulation (GeneJammer®) for mesenchymal stem cells (MSCs).[29] Nonviral gene transfer approaches are commonly employed for the ex vivo modification of cells [30] as their efficacy (~35–40%) is lower than those achieved with viral vectors and in light of the lack of success to modify chondrocytes directly in the cartilaginous ECM.
4.2. Viral vectors Vectors based on adenovirus allow for high in vitro gene transfer efficiencies and expression levels both in chondrocytes (between 90% and close to 100%) [31– 33] and in MSCs (up to 80%),[33] but their use in vivo leads to host immune responses while transgene expression remains brief (1 – 2 weeks), an issue to treat a progressive disease like OA. Of further note, intra-articular injection of such vectors mostly targets the synovial cells, without proper access to the articular cartilage.[34] Retroviral vectors are attractive carriers as they have an ability to stably integrate in the host genome for persistence of the transgene delivered. A drawback of this class of vectors is the possibility to activate tumor genes and to provoke insertional mutagenesis events upon integration. While employed to modify other cells and tissues, retroviral vectors may not be best suited to target quiescent chondrocytes as these vehicles only transduce dividing cells, allowing for relatively low efficiencies (
