Spine update: potential applications of gene therapy to the treatment of spinal disorders.
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Biomedical subjects
C H Evans
Publications and source records attributed to C H Evans.
GAG-augmented polysaccharide hydrogel: a novel biocompatible and biodegradable material to support chondrogenesis.
The quality of articular cartilage engineered using a cell-polymer construct depends, in part, on the chemical composition of the biomaterial and whether that biomaterial can support the chondrocytic phenotype. Acknowledging the supportive influence of tissue-specific matrix molecules on the chondrocytic phenotype, we have combined chondroitin sulfate-A (CSA) and chitosan, a glycosaminoglycan (GAG) analog, to develop a novel biomaterial to support chondrogenesis. Chitosan is a polycationic repeating monosaccharide of beta-1,4-linked glucosamine monomers with randomly located N-acetyl glucosamine units. Chitosan may be combined with the polyanionic CSA such that ionic crosslinking results in hydrogel formation. Bovine primary articular chondrocytes, when seeded onto a thin layer of CSA-chitosan, form discrete, focal adhesions to the material and maintain many characteristics of the differentiated chondrocytic phenotype, including round morphology, limited mitosis, collagen type II, and proteoglycan production. Our findings suggest CSA-chitosan may be well suited as a carrier material for the transplant of autologous chondrocytes or as a scaffold for the tissue engineering of cartilage-like tissue.
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Tumor necrosis factor-alpha promotes human papillomavirus (HPV) E6/E7 RNA expression and cyclin-dependent kinase activity in HPV-immortalized keratinocytes by a ras-dependent pathway.
Tumor necrosis factor-alpha (TNF-alpha) inhibits growth of normal cervical keratinocytes but stimulates proliferation of human papillomavirus (HPV)-immortalized and cervical carcinoma-derived cell lines when mitogens such as epidermal growth factor (EGF) or serum are depleted. Current work identifies the mechanism of growth stimulation. TNF-alpha promoted cell cycle progression by increasing expression of HPV-16 E6/E7 RNAs and enhancing activity of cyclin-dependent kinase (cdk)2 and cdc2 after 3 d. Increased kinase activity was mediated by upregulation of cyclins A and B and decreases in cdk inhibitors p21(waf) and p27(kip). TNF-alpha stimulated these changes in part by increasing transcription and stabilization of RNA for amphiregulin, an EGF receptor ligand, and amphiregulin directly increased HPV-16 E6/E7 and cyclin A RNAs. To define which components of the EGF receptor signaling pathway were important, HPV-immortalized cells were transfected with activated or dominant negative mutants of Ha-ras, raf, or MAPKK. Expression of activated Ha-ras maintained HPV-16 and cyclin gene expression and promoted rapid growth in the absence of EGF. Furthermore, ras activation was necessary for TNF-alpha mitogenesis as transfection with a dominant negative ras mutant (Asn-17) strongly inhibited growth. Thus, activation of ras promotes expression of HPV-16 E6/E7 RNAs, induces cyclins A and B, and mediates growth stimulation of immortal keratinocytes by TNF-alpha. These studies define a pathway by which ras mutations, which occur in a subset of cervical cancers, may contribute to pathogenesis. Mol. Carcinog. 27:97-109, 2000. Published by Wiley-Liss, Inc.
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Genetic enhancement of matrix synthesis by articular chondrocytes: comparison of different growth factor genes in the presence and absence of interleukin-1.
OBJECTIVE: To determine whether articular chondrocytes express growth factor genes delivered by adenoviral vectors and whether expression of these genes influences matrix synthesis in the presence and absence of interleukin-1 (IL-1). METHODS: Monolayer cultures of rabbit articular chondrocytes were infected with recombinant adenovirus carrying genes encoding the following growth factors: insulin-like growth factor 1 (IGF-1), transforming growth factor beta1 (TGFbeta1), and bone morphogenetic protein 2 (BMP-2). As a control, cells were transduced with the lac Z gene. Cultures were also treated with each growth factor supplied as a protein. Levels of gene expression were noted, and the synthesis of proteoglycan, collagen, and noncollagenous proteins was measured by radiolabeling. Collagen was typed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The effects of growth factor gene transfer on proteoglycan synthesis in the presence of IL-1 were also measured. RESULTS: The expression of all transgenes was high following adenoviral transduction. Proteoglycan synthesis was stimulated approximately 8-fold by the BMP-2 gene and 2-3-fold by the IGF-1 gene. The effects of BMP-2 and IGF-1 genes were additive upon cotransduction. Synthesis of collagen and noncollagenous proteins, in contrast, was most strongly stimulated by the IGF-1 gene. In each case, collagen typing confirmed the synthesis of type II collagen. IL-1 suppressed proteoglycan synthesis by 50-60%. IGF-1 and TGFbeta genes restored proteoglycan synthesis to control levels in the presence of IL-1. The BMP-2 gene, in contrast, elevated proteoglycan synthesis beyond control levels in the presence of IL-1. CONCLUSION: Transfer of growth factor genes to articular chondrocytes can greatly increase matrix synthesis in vitro, even in the presence of the inflammatory cytokine IL-1. This result encourages the further development of gene therapy for the repair of damaged cartilage.
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Adenovirus-mediated gene transfer of insulin-like growth factor 1 stimulates proteoglycan synthesis in rabbit joints.
OBJECTIVE: To examine the effect of insulin-like growth factor 1 (IGF-1) on the regulation of cartilage synthesis and other articular events in vivo. METHODS: A first-generation adenoviral vector expressing human IGF-1 (AdIGF-1) from the cytomegalovirus promoter was constructed. Particles of AdIGF-1 (5 x 10(9)) were injected through the patellar tendon into normal rabbit knee joints and rabbit knee joints with antigen-induced arthritis (AIA), with the same dose of a control adenoviral vector injected into the contralateral knees. Lavage fluids were obtained from rabbit knee joints on days 3 and 7 postinjection and used for analysis of IGF-1 expression, white blood cell infiltration, and cartilage breakdown. Cartilage chips from rabbit joints were used for assay of new proteoglycan synthesis, and tissues also were harvested from the dissected knees for histologic study. RESULTS: Intraarticular injection of AdIGF-1 resulted in a mean of 180.6 ng/ml of IGF-1 expression in the lavage fluid from rabbit joints. IGF-1 expression stimulated new proteoglycan synthesis in both naive and AIA rabbit knees, but had no significant chondroprotective or antiinflammatory effects. Histologic analysis showed that elevated levels of IGF-1 expression in both normal and arthritic knees had no adverse pathologic effects on synovium or adjacent muscles. CONCLUSION: Gene transfer of IGF-1 into rabbit knee joints promotes proteoglycan synthesis without significantly affecting inflammation or cartilage breakdown. In addition, no adverse effects following intraarticular IGF-1 gene delivery were observed. Thus, local gene transfer of IGF-1 to joints could serve as a therapeutic strategy to stimulate new matrix synthesis in both rheumatoid arthritis and osteoarthritis.
Adenoviridae↗
Increased matrix synthesis following adenoviral transfer of a transforming growth factor beta1 gene into articular chondrocytes.
Monolayer cultures of lapine articular chondrocytes were transduced with first-generation adenoviral vectors carrying lacZ or transforming growth factor beta1 genes under the transcriptional control of the human cytomegalovirus early promoter. High concentrations of transforming growth factor beta1 were produced by chondrocytes following transfer of the transforming growth factor beta1 gene but not the lacZ gene. Transduced chondrocytes responded to the elevated endogenous production of transforming growth factor beta1 by increasing their synthesis of proteoglycan, collagen, and noncollagenous proteins in a dose-dependent fashion. The increases in collagen synthesis were not accompanied by alterations in the collagen phenotype; type-II collagen remained the predominant collagen. Transforming growth factor beta1 could not, however, rescue the collagen phenotype of cells that had undergone phenotypic modulation as a result of serial passaging. These data demonstrate that chondrocytes can be genetically manipulated to produce and respond to the potentially therapeutic cytokine transforming growth factor beta1. This technology has a number of experimental and therapeutic applications, including those related to the study and treatment of arthritis and cartilage repair.
Adenoviridae↗
[TGF-beta-1 gene transfer in joint cartilage cells. Stimulating effect in extracellular matrix synthesis].
TGF beta-1 has been shown to upregulate matrix synthesis in articular chondrocytes. TGF beta-gene transfer to chondrocytes has the potential to increase the local production of this key component within regenerating cartilage after trauma and could support the repair process in articular cartilage lesions. Primary rabbit articular chondrocytes were cultured and retrovirally transfected with the experimental TGF beta-1 and the lacZ marker gene for control purposes. After radioactive labeling of new synthesized matrix proteins results were compared with normal primary chondrocytes. After TGF beta-1 gene transfer the endogenous growth factor concentration was doubled compared to normal chondrocytes and decreased in the lacZ control group. The proteoglycan synthesis in TGF beta-1 transfected chondrocytes showed a 96% increase compared to the basal production of normal chondrocytes. The LacZ transfected group revealed the opposite effect by a 44% decrease. The collagen synthesis of TGF beta-1 transfected chondrocytes was 304% compared to normal chondrocytes, predominantly type II collagen. The lacZ group collagen production was reduced by 35%. We conclude that TGF beta-1 gene transfer overcomes the decreasing effect observed by transfection with the LacZ marker gene and increases matrix synthesis in articular chondrocytes. Genetically altered chondrocytes might improve the repair of cartilage lesions by stimulating matrix synthesis and supporting the expression of the hyaline phenotype.
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[Current aspects of tendon healing].
This review describes structure, function and healing of tendinous tissue and discusses new biologically based treatment options to modulate tendon healing. The repair process after tendon rupture results in a morphologically different and biomechanically inferior structure compared to a normal tendon. The collagen fibril diameters are decreased months after the traumatic lesion and show also different phenotypes. We know that cytokines and growth factors are key components for normal tissue development and regulate wound healing processes. Some growth factors have been detected to influence tenocytes by promoting cell proliferation and matrix synthesis. Application of the adequate growth factors at certain periods during the repair process might improve the healing result after tendon rupture. However, most of these growth factors are proteins which are rapidly metabolized by the organism. Transfer of growth factor genes into tenocytes might eliminate this problem by a continuous local release of growth factors at the healing site.
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Gene therapy for autoimmune disorders.
Although many autoimmune disorders do not have a strong genetic basis, their treatment may nevertheless be improved by gene therapies. Most strategies seek to transfer genes encoding immunomodulatory products that will alter host immune responses in a beneficial manner. Used in this fashion, genes serve as biological delivery vehicles for the products they encode. By this means gene therapy overcomes obstacles to the targeted delivery of proteins and RNA, and improves their efficacy while providing a longer duration of effect, and, potentially, greater safety. Additional genetic strategies include DNA vaccination and the ablation of selected tissues and cell populations. There is considerable evidence from animal studies that gene therapies work: examples include the treatment of experimental models of rheumatoid arthritis, multiple sclerosis, diabetes, and lupus. Pre-clinical success in treating animal models of rheumatoid arthritis has led to the first clinical trial of gene therapy for an autoimmune disease. In this Phase I study, a cDNA encoding the interleukin-1 receptor antagonist was transferred to the knuckle joints of patients with advanced rheumatoid arthritis. Two additional clinical trials are in progress. It is likely that gene therapy will provide effective new treatments for a wide range of autoimmune disorders.
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Genetic enhancement of fracture repair: healing of an experimental segmental defect by adenoviral transfer of the BMP-2 gene.
This study evaluated the ability of gene transfer to enhance bone healing. Segmental defects were created surgically in the femora of New Zealand white rabbits. First generation adenoviruses were used as vectors to introduce into the defects genes encoding either human bone morphogenetic protein-2 (BMP-2) or, as a negative control, firefly luciferase. Representative specimens were evaluated histologically after 8 weeks. Healing of the defects was monitored radiographically for 12 weeks, after which time the repair tissue was evaluated biomechanically. By radiological criteria, animals receiving the BMP-2 gene had healed their osseous lesions after 7 weeks, whereas those receiving the luciferase gene had not. Histologic examination of representative rabbits at 8 weeks confirmed ossification across the entire defect in response to the BMP-2 gene, whereas the control defect was predominantly fibrotic and sparsely ossified. At the end of the 12-week experiment, the control femora still showed no radiological signs of stable healing. The difference in radiologically defined healing between the experimental and control groups was statistically significant (P < 0. 002). Biomechanical testing of the femora at 12 weeks demonstrated statistically significant increases in the mean bending strength (P < 0.005) and bending stiffness (P < 0.05) of the animals treated with the BMP-2 gene. Direct, local adenoviral delivery of an osteogenic gene thus led to the healing of an osseous lesion that otherwise would not do so. These promising data encourage the further development of genetic approaches to enhancing bone healing. Gene Therapy (2000) 7, 734-739.
Adenoviridae↗
Gene therapy for meniscal injury: enhanced synthesis of proteoglycan and collagen by meniscal cells transduced with a TGFbeta(1)gene.
Objective To determine whether meniscal cells can express a TGFbeta(1)transgene delivered by a retroviral vector, and respond to the gene product by increasing matrix synthesis. Methods Monolayer cultures of human and canine meniscal cells were infected with retroviruses carrying either a human TGFbeta(1)cDNA or marker genes. Conditioned media were assayed for the presence of TGFbeta(1). Biosynthesis assays using radiolabeled precursors were employed to determine the effects of the transgenes on the synthesis of proteoglycan, collagen and noncollagenous proteins. Collagen phenotyping was performed by SDS-PAGE. Results Media conditioned by canine and human meniscal cells transduced with the TGFbeta(1)gene, accumulated several nanograms/10(6)cells of TGFbeta(1)during a 48 h incubation. Media conditioned by control cells contained very little TGFbeta(1). Transduction with the TGFbeta(1)gene, but not marker genes, increased the synthesis of collagen and proteoglycan by 8-15-fold. The synthesis of noncollagenous proteins was enhanced more modestly. Monolayers of meniscal cells synthesized types I, III, V and VI collagen. The TGFbeta(1)gene increased the synthesis of all types of collagen without altering the ratios between them. Conclusions Meniscal cells are readily transduced by retroviral vectors and respond to the transfer of a TGFbeta(1)cDNA by greatly increasing matrix synthesis. These findings encourage the further development of genetic approaches to the healing of meniscal lesions.
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Potential role of direct adenoviral gene transfer in enhancing fracture repair.
Gene therapy has much to offer in the treatment of conditions in which it is necessary to increase the formation of bone. Nonunions, segmental defects, and aseptic loosening are examples of conditions where the local expression of genes that inhibit osteolysis and promote osteogenesis might be helpful. Studies in which one such possibility has been evaluated experimentally are described. These investigations used a surgically produced segmental defect in the femurs of New Zealand White rabbits as the model system. Adjacent muscle was fashioned around the defect to form a chamber into which adenoviral vectors were injected. High levels of transgene expression were found in the muscle surrounding the defect after injection of vectors carrying marker genes. Transgene expression also was seen in the cut ends of the bone and the scar tissue within the gap. No transgene expression was seen in the contralateral limb, spleen, or lung; transient, low levels of expression were found in the liver. Transgene expression declined with time, disappearing from all tissue but bone by Day 26; expression persisted in bone for at least 6 weeks. The control defects did not heal spontaneously. Injection of adenovirus carrying a human bone morphogenetic protein-2 complementary deoxyribonucleic acid led to healing of the segmental defect within 12 weeks, as judged by radiographic, histologic, and biomechanical criteria. Adenovirus carrying a human transforming growth factor-beta 1 complementary deoxyribonucleic acid showed signs of improved healing, but not to the extent seen with the bone morphogenetic protein-2 complementary deoxyribonucleic acid. This approach to therapy holds much promise as a novel means of promoting osteogenesis.
Adenoviridae↗
Insulinlike growth factor-I gene therapy applications for cartilage repair.
Cartilage function after resurfacing with cell-based transplantation procedures or during the early stages of arthritic disease may be bolstered by the addition of growth factor genes to the transplanted tissue. Insulinlike growth factor-I maintains chondrocyte metabolism in normal cartilage homeostasis and has been shown to improve cartilage healing in vivo. Given the relatively short half-life of insulinlike growth factor-I in biologic systems, however, maintenance of effective concentrations of this peptide has necessitated either very high initial doses or repeated treatment. Delivery of the insulinlike growth factor-I gene, using a deleted adenovirus vector, specifically targeting graftable articular chondrocytes, bone marrow-derived chondroprogenitor cells, or synovial lining cells, may provide more durable insulinlike growth factor-I fluxes to articular tissues. Cultured equine articular chondrocytes, mesenchymal stem cells, synovial explants, and synovial intimal cells were readily transfected with an E1-deleted adenoviral vector containing equine insulinlike growth factor-I coding sequence. Optimal viral concentrations for effective transduction were 100 multiplicities of infection in synoviocytes, 500 multiplicities of infection in chondrocytes, and 1000 multiplicities of infection in mesenchymal stem cells. Production of insulinlike growth factor-I ligand varied from 65 ng/mL to 246 ng/mL in medium from chondrocytes and synovial explants, respectively. For chondrocytes, these concentrations were sufficient to produce significant stimulation of cartilage matrix gene expression and subsequent proteoglycan production. Moreover, cells in infected cultures maintained a chondrocytic phenotype and continued to express elevated insulinlike growth factor-I levels during 28 days of monolayer culture. Minimal synthetic activity, other than insulinlike growth factor-I ligand synthesis, was evident in synovial cultures. These experiments suggest several avenues for insulinlike growth factor-I supplementation of articular cartilage, including preimplantation adenoviral-insulinlike growth factor gene transfer to chondrocytes or chondroprogenitor cells, and direct injection of adenoviral-insulinlike growth factor to transfect the synovial structures in situ.
Adenoviridae↗
Using gene therapy to protect and restore cartilage.
Numerous gene products have the potential to help protect cartilage from degradation and to repair cartilage that has become damaged as a result of disease or injury. The genes that encode these products thus may serve as chondroprotective and chondroregenerative medicines. To bring these agents into clinical use, it is necessary to screen candidate genes for efficacy under in vitro and in vivo conditions, to determine the best cells to target, and to develop appropriate gene transfer technologies. As discussed in the current review, progress has been made in each of these areas. Various viral and nonviral vectors are able to deliver genes to synoviocytes, articular chondrocytes, and mesenchymal stem cells. There also is evidence to suggest that ex vivo and in vivo approaches can be used for gene transfer to articular cartilage, synovium, and meniscus. Moreover various cytokine antagonists and growth factors have been shown to protect cartilage and stimulate chondrogenesis. In vivo methods and strategies that target synovium may be useful in a chondroprotective mode but because they do not increase the number of chondrogenic cells within lesions, they may be ill-equipped to repair large defects. Ex vivo methods however, provide cells and genes. It also is important to distinguish the treatment of isolated lesions occurring as a result of injury from the treatment of lesions resulting from underlying disease processes. Additional development of these approaches should result in clinically useful genetic methods for the protection and regeneration of cartilagenous tissues.
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Potential applications of gene therapy to the treatment of intervertebral disc disorders.
Gene therapy involves the transfer of genes to cells such that the recipient cells express these genes and thereby synthesize the ribonucleic acid and protein that they encode. Recent investigations suggest that gene therapy may have potential applications in the treatment of intervertebral disc disorders, particularly those associated with disc degeneration. The successful in vivo transfer of therapeutic genes to target cells within the intervertebral disc in clinically relevant animal models is one example of the rapid progress that is being made. The purpose of the current review is to address several important technical issues, including choice of vectors and gene delivery strategy and the characteristics of the target tissues, which are relevant to future clinical applications of gene therapy for the treatment of intervertebral disc disorders. It already is apparent from the growing literature that gene therapy has the potential of becoming a valuable clinical treatment mode for intervertebral disc disorders in the twenty-first century.
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Gene therapy approaches for treating rheumatoid arthritis.
Current gene therapy approaches for treating rheumatoid arthritis have made use of gene transfer technology as an improved delivery system for emerging proteins and other biologicals whose activities may have therapeutic value. Preclinical research has focused on two primary directions, evaluation of methods of gene delivery and identification of gene products with antiarthritic potential. Although there are reports involving systemic gene delivery, the bulk of effort has focused on local, intraarticular administration using ex vivo and in vivo methods. Viral-based vectors, including adenovirus, adeno-associated virus and herpes simplex virus have the greatest efficiency of gene delivery after intraarticular injection and are capable of generating relevant levels of gene products in several animal models of disease. However, there are limitations to existing generations of these systems that currently preclude their clinical application. Those gene products found to be efficacious in animal models of rheumatoid arthritis include proteins that specifically block the activity of the primary inflammatory cytokines, and include interleukin-1 receptor antagonist and soluble receptors for tumor necrosis factor and interleukin-1. Delivery and expression of genes encoding certain cytokines such as interleukins -4, -10, and -13 and viral interleukin-10, that block synthesis of inflammatory mediators and downregulate aspects of cellular and humoral immune pathways have been found beneficial. Although significant progress has been made, leading to Phase I clinical trials, there remain several hurdles to the routine practice of gene therapy for treatment of rheumatoid arthritis.
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Clinical trials in the gene therapy of arthritis.
Gene therapy clinical trials raise important safety issues that complicate their design and require extensive preclinical testing. Human protocols for the treatment of arthritis and most other orthopaedic and rheumatologic indications are complicated additionally by the perception that they are largely acquired, nonlethal conditions. Taking these considerations into account, the first such human study used the local, ex vivo delivery of a gene whose product, the interleukin-1 receptor antagonist, has an outstanding safety profile. This gene was delivered to the metacarpophalangeal joints of postmenopausal women 1 week before these joints were removed during total joint replacement surgery. In addition to providing an additional safety cushion, the surgical removal of the genetically modified joints made available large amounts of tissue to examine for evidence of successful gene transfer and gene expression. This Phase I safety study was approved at the local and federal levels, and its funding was contingent on the establishment of an external monitoring board. This trial now has been completed and a Phase II, efficacy study is being planned. A similar study has begun in Dusseldorf, Germany and results from the first two patients are similar to the results of the American patients. Permission has been given for two additional human trials, one in the United States and one in the Netherlands, in which a gene encoding herpes thymidine kinase will be transferred to the joints of patients with rheumatoid arthritis who then will be administered gancyclovir. This procedure aims to treat the disease by producing a genetic synovectomy. Additional development of human gene therapies for arthritis and other orthopaedic and rheumatic conditions will be aided by the successful completion of these studies.
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Gene therapy for rheumatoid arthritis.
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