Julio C. Fernandes

Chitosan-DNA nanoparticles as non-viral vectors in gene therapy: strategies to improve transfection efficacy.

Currently, the major drawback of gene therapy is the gene transfection rate. The two main types of vectors that are used in gene therapy are based on viral or non-viral gene delivery systems. The viral gene delivery system shows a high transfection yield but it has many disadvantages, such as oncogenic effects and immunogenicity. However, cationic polymers, like chitosan, have potential for DNA complexation and may be useful as non-viral vectors for gene therapy applications. Chitosan is a natural non-toxic polysaccharide, it is biodegradable and biocompatible, and protects DNA against DNase degradation and leads to its condensation. The objective of this paper was to summarize the state of the art in gene therapy and particularly the use of chitosan to improve the transfection efficiency in vivo and in vitro.

Reduced progression of experimental osteoarthritis in vivo by selective inhibition of inducible nitric oxide synthase

OBJECTIVE To evaluate the in vivo therapeutic efficacy of N-iminoethyl-L-lysine (L-NIL), a selective inhibitor of inducible nitric oxide synthase (iNOS), on the progression of lesions in an experimental osteoarthritis (OA) dog model. The effect of L-NIL on metalloprotease activity, levels of interleukin-1beta (IL-1beta), prostaglandin E2 (PGE2), and nitrite/nitrate in synovial fluid was determined. METHODS The OA model was created by sectioning the anterior cruciate ligament of the right stifle joint of mongrel dogs by a stab wound. Dogs were separated into experimental groups: Group 1 was made up of unoperated dogs that received no treatment, group 2 were operated dogs with no treatment, and group 3 were operated dogs that received oral L-NIL (10 mg/kg/twice daily) starting immediately after surgery. The OA dogs were killed at 10 weeks after surgery. RESULTS Experiments showed that dog OA cartilage explants in culture produced an increased amount of NO (nitrite). Immunohistochemical study demonstrated that this was due to an increased level of iNOS in chondrocytes. OA dogs treated with L-NIL showed a reduction in the incidence of osteophytes compared with the untreated OA dogs (58% versus 92%) as well as in their size (mean +/- SEM 1.92 +/- 0.58 mm versus 5.08 +/- 0.66 mm). Macroscopically, L-NIL decreased the size of the cartilage lesions by approximately 50% both on condyles and plateaus. The histologic severity of both the cartilage lesions and synovial inflammation was significantly decreased in the L-NIL-treated dogs. Treatment with L-NIL also significantly decreased both collagenase and general metalloprotease activity in the cartilage and the levels of IL-1beta, PGE2, and nitrite/nitrate in synovial fluid. CONCLUSION This study demonstrated the effectiveness of a selective inhibitor of iNOS, L-NIL, in attenuating the progression of experimental OA. The data suggest that L-NIL may act by reducing the activity of metalloproteases in cartilage and the production of IL-1beta by synovium, both of which are known to play a major role in the pathophysiology of OA structural changes.

Mesenchymal stem cells, MG63 and HEK293 transfection using chitosan-DNA nanoparticles

Chitosan-DNA nanoparticles were synthesized from the complexation of the cationic polymer with a ss-gal DNA plasmid, in order to study the efficacy of chitosan to develop a non-viral gene delivery system that can be optimized for efficient gene therapy. The optimal binding conditions were determined with the fluorescamine and PicoGreen assays. DNA distribution within the nanoparticle was visualized by electron transmission microscopy, while the size and morphology were assessed by atomic force microscopy. The transfection potential was evaluated for the first time on human mesenchymal stem cells (MSCs), on human osteosarcoma cells (MG63) and on human embryonic kidney cells (HEK293). The LipofectAMINE(TM) 2000 (LF) reagent was used in comparison. The effect of chitosan-DNA nanoparticles on cell viability was illustrated with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The nanoparticles formed are of a diameter inferior to 100nm with a homogenous distribution of DNA. The transfection of HEK293 cells is superior to that seen with MG63 cells and MSCs, however not surpassing that seen with LF. Minimal cytotoxicity is seen with the polyplexes compared to greater than 50% toxicity with LF. These results suggest that chitosan-DNA nanoparticles have favorable characteristics for non-viral gene delivery, are cell type dependent and not cytotoxic.

In Vivo Transfer of Interleukin-1 Receptor Antagonist Gene in Osteoarthritic Rabbit Knee Joints: Prevention of Osteoarthritis Progression

The goal of this study was to determine the efficacy of local IL-1Ra gene therapy by intra-articular plasmid injections on structural changes in the meniscectomy rabbit model of osteoarthritis. A partial meniscectomy of the right knee was performed on the rabbits through a medial parapatellar incision. The rabbits were then divided into four experimental groups. Group 1 received no treatment. Group 2 received three consecutive intra-articular injections at 24-hour intervals of 0.9% saline containing a lipid, gammaAP-DLRIE/DOPE, and a DNA plasmid, VR1012. Group 3 received three consecutive injections of saline containing 1000 microg of canine IL-1Ra plasmid and lipid. The injections were given starting 4 weeks post-surgery. Rabbits from Group 1 were killed 4 weeks post-surgery, and all other rabbits 8 weeks post-surgery. The severity of macroscopic and microscopic changes on cartilage on the medial and femoral condyles and tibial plateaus and synovium were graded separately. Specimens were also processed for immunohistochemical staining using a rabbit polyclonal antibody against canine IL-1Ra. The level of canine IL-1Ra in synovial fluid was determined using enzyme-linked immunosorbent assay. The presence of the DNA plasmid in the synovium was tested by polymerase chain reaction. A significant reduction in the width of osteophytes and size of macroscopic lesions (P < 0.04) was observed, and was dependent on the amount of IL-1Ra plasmid injected. A significant reduction was also noted in the severity of histologic cartilage lesions (P < 0.01) in the group that received the highest dosage (1000 microg) of IL-1Ra plasmid. IL-1Ra was detected in synovial fluid by enzyme-linked immunosorbent assay and by immunohistochemical staining in the synovium and cartilage of rabbits that received injections containing the IL-1Ra plasmid. Polymerase chain reaction analysis of synovial DNA revealed the presence of the cloned cDNA dog IL-1Ra up to 4 weeks after the first intra-articular injection. This study demonstrates that direct in vivo transfer of the IL-1Ra gene into osteoarthritis knee cells using intra-articular injections of a plasmid vector and lipids can significantly reduce the progression of experimental osteoarthritis. This avenue may therefore represent a promising future treatment for osteoarthritis.

Selective inhibition of inducible nitric oxide synthase reduces progression of experimental osteoarthritis in vivo: Possible link with the reduction in chondrocyte apoptosis and caspase 3 level

OBJECTIVE To evaluate the in vivo therapeutic efficacy of N-iminoethyl-L-lysine (L-NIL), a selective inhibitor of inducible nitric oxide synthase, on the progression of structural lesions in the experimental canine model of osteoarthritis (OA), and to explore the effect of L-NIL on the level of chondrocyte apoptosis and of important proteins involved in the apoptotic phenomenon, i.e., caspase 3 (inducer) and Bcl-2 (inhibitor). METHODS The OA model was created by sectioning the anterior cruciate ligament. Dogs were placed into 4 experimental groups: unoperated dogs that received no treatment (controls), operated (OA) dogs that received placebo treatment, OA dogs that received oral L-NIL at 10 mg/kg/day, and OA dogs that received oral L-NIL at 1.0 mg/kg/day. In both L-NIL groups, treatment started immediately after surgery. The OA dogs were killed at 12 weeks after surgery. RESULTS OA dogs treated with L-NIL showed a reduction in the size of osteophytes and a significant decrease in the severity of macroscopic and histologic cartilage lesions on both condyles and plateaus, compared with untreated OA dogs. L-NIL treatment also significantly decreased metalloprotease activity in cartilage. Immunohistochemical analysis revealed that the levels of chondrocyte apoptosis, caspase 3, and Bcl-2 were markedly increased in OA cartilage (P < 0.0001). A positive correlation between the levels of chondrocyte apoptosis and levels of caspase 3 was found (r = 0.54, P < 0.0001). OA dogs treated with the higher dosage L-NIL showed significantly reduced levels of chondrocyte apoptosis (P < 0.003) and caspase 3 (P < 0.04), but no effect on the increased level of Bcl-2 was demonstrated. CONCLUSION This study shows that L-NIL reduces the progression of experimental OA. This effect could be related to a reduced level of chondrocyte apoptosis and is likely mediated by a decrease in the level of caspase 3 activity. A sparing effect of L-NIL on the increased level of Bcl-2 may also be a contributing factor.

Uptake mechanisms of non-viral gene delivery.

Non-viral gene delivery is currently a hot subject for its relative safety and simplicity of use; however, it is still far from being ideal enough to be clinically used for its comparatively lower efficiency than viral gene delivery. To improve the efficiency of non-viral gene delivery needs a comprehensive understanding of the uptake mechanisms. Macromolecules are internalized into cells by a variety of mechanisms, and their intracellular fates are usually relevant with the uptake pathways. The uptake pathways of non-viral gene complexes are usually determined by not only the gene/carrier interaction but also by the interaction between complexes and target cells. The best-characterized uptake pathway is the so-called clathrin-mediated endocytic pathway. However, there are numerous updates of knowledge about endocytic pathways and even non-endocytic pathways in recent years with the development of novel technologies for tracking and inhibiting. In this review, we will try to sort out our current understanding of the uptake mechanisms of non-viral gene delivery. In addition, factors for pathway selection are summarized in the third section. Finally, the useful inhibitors or tools for the study of these pathways will also be concluded in the last section.

In vivo dual inhibition of cyclooxygenase and lipoxygenase by ML-3000 reduces the progression of experimental osteoarthritis: Suppression of collagenase 1 and interleukin-1β synthesis

OBJECTIVE To study the therapeutic effectiveness of ML-3000, a new antiinflammatory drug that has balanced dual inhibitory activity against 5-lipoxygenase and cyclooxygenase, on the development of lesions in the experimental osteoarthritis (OA) dog model, and to determine the action of ML-3000 on the synthesis of collagenase 1 in cartilage and interleukin-1beta (IL-1beta) in synovial membrane. METHODS The anterior cruciate ligament of the right stifle joint of 21 mongrel dogs was sectioned with a stab wound. Dogs were divided into 3 groups: group 1 (n = 7) received placebo; groups 2 (n = 7) and 3 (n = 7) were treated with therapeutic dosages of oral ML-3000 at 2.5 mg/kg/day and 5 mg/kg/day, respectively. The dogs began receiving medication the day after surgery and were killed 8 weeks later. The size and grade of cartilage erosions on both the condyles and plateaus were evaluated, and the severity of the cartilage lesions and synovial inflammation was examined histologically. Levels of collagenase 1 in cartilage and IL-1beta in the synovial membrane were measured by immunohistochemistry. In addition, levels of prostaglandin E2 (PGE2) in the synovial fluid and leukotriene B4 (LTB4) in cultured synovial membrane explants were determined using specific enzyme immunoassays. RESULTS Serum levels of ML-3000 in treated dogs were within the therapeutic range. ML-3000 significantly decreased the size and grade of the cartilage lesions in tibials and plateaus, compared with placebo. At the histologic level, the severity of cartilage lesions was also decreased in the ML-3000-treated dogs versus the placebo-treated dogs in both the condyles and the plateaus. All 3 OA groups exhibited a notable and similar level of synovial inflammation. ML-3000 significantly decreased the level of PGE2 in synovial fluid and LTB4 production by synovium. It also markedly reduced the levels of collagenase 1 in cartilage and IL-1beta in synovial membrane. CONCLUSION ML-3000 significantly reduced the development of lesions in experimental dog OA. The drug acts by reducing the synthesis of the inflammation mediators PGE2 and LTB4 and catabolic factors such as collagenase 1 and IL-1beta, which are known to play an important role in the pathophysiology of OA lesions. The effect of the drug on catabolic factors could possibly be related to its inhibitory action on LTB4 synthesis.

Polycation-Based Gene Therapy: Current Knowledge and New Perspectives

At present, gene transfection insufficient efficiency is a major drawback of non-viral gene therapy. The 2 main types of delivery systems deployed in gene therapy are based on viral or non-viral gene carriers. Several non-viral modalities can transfer foreign genetic material into the human body. To do so, polycation-based gene delivery methods must achieve sufficient efficiency in the transportation of therapeutic genes across various extracellular and intracellular barriers. These barriers include interactions with blood components, vascular endothelial cells and uptake by the reticuloendothelial system. Furthermore, the degradation of therapeutic DNA by serum nucleases is a potential obstacle for functional delivery to target cells. Cationic polymers constitute one of the most promising approaches to the use of viral vectors for gene therapy. A better understanding of the mechanisms by which DNA can escape from endosomes and traffic to enter the nucleus has triggered new strategies of synthesis and has revitalized research into new polycation-based systems. The objective of this review is to address the state of the art in gene therapy with synthetic and natural polycations and the latest advances to improve gene transfer efficiency in cells.

Synthetic and Natural Polycations for Gene Therapy: State of the Art and New Perspectives

Currently, the major drawback of gene therapy is the gene transfection rate. The two main types of vectors that are used in gene therapy are based on viral or non-viral gene delivery systems. There are several non-viral systems that can be used to transfer foreign genetic material into the human body. In order to do so, the DNA to be transferred must escape the processes that affect the disposition of macromolecules. These processes include the interaction with blood components, vascular endothelial cells and uptake by the reticuloendothelial system. Furthermore, the degradation of therapeutic DNA by serum nucleases is also a potential obstacle for functional delivery to the target cell. Cationic polymers have a great potential for DNA complexation and may be useful as non-viral vectors for gene therapy applications. The objective of this review was to address the state of the art in gene therapy using synthetic and natural polycations and the latest strategies to improve the efficiency of gene transfer into the cell.

4-Hydroxynonenal induces apoptosis in human osteoarthritic chondrocytes: the protective role of glutathione-S-transferase

Introduction4-Hydroxynonenal (HNE) is one of the most abundant and reactive aldehydes of lipid peroxidation products and exerts various effects on intracellular and extracellular signalling cascades. We have previously shown that HNE at low concentrations could be considered as an important mediator of catabolic and inflammatory processes in osteoarthritis (OA). In the present study, we focused on characterizing the signalling cascade induced by high HNE concentration involved in cell death in human OA chondrocytes.MethodsMarkers of apoptosis were quantified with commercial kits. Protein levels were evaluated by Western blotting. Glutathione (GSH) and ATP levels were measured with commercial kits. Glucose uptake was assessed by 2-deoxy-D-[3H]-glucose. The role of GSH-S-transferase A4-4 (GSTA4-4) in controlling HNE-induced chondrocyte apoptosis was investigated by chondrocyte transfection with small interfering RNA (siRNA) or with the expression vector of GSTA4-4.ResultsOur data showed that HNE at concentrations of up to 10 μM did not alter cell viability but was cytotoxic at concentrations of greater than or equal to 20 μM. HNE-induced chondrocyte death exhibited several classical hallmarks of apoptosis, including caspase activation, cytochrome c and apoptosis-induced factor release from mitochondria, poly (ADP-ribose) polymerase cleavage, Bcl-2 downregulation, Bax upregulation, and DNA fragmentation. Our study of signalling pathways revealed that HNE suppressed pro-survival Akt kinase activity but, in contrast, induced Fas/CD95 and p53 expression in chondrocytes. All of these effects were inhibited by an antioxidant, N-acetyl-cysteine. Analysis of cellular energy and redox status showed that HNE induced ATP, NADPH, and GSH depletion and inhibited glucose uptake and citric acid cycle activity. GSTA4-4 ablation by the siRNA method augmented HNE cytotoxicity, but, conversely, its overexpression efficiently protected chondrocytes from HNE-induced cell death.ConclusionOur study provides novel insights into the potential mechanisms of cell death in OA cartilage and suggests the potential role of HNE in OA pathophysiology. GSTA4-4 expression is critically important for cellular defence against oxidative stress-induced cell death in OA cartilage, possibly by HNE elimination.