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.

Intra-articular adenoviral-mediated gene transfer of trail induces apoptosis of arthritic rabbit synovium.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that primarily affects joints. In rheumatoid joints there is extensive synovial proliferation with diseased synovium becoming highly aggressive, attaching to the articular cartilage and bone to form what is termed a pannus. The formation of active pannus is central to erosive disease and resulting joint destruction. In this study, we examined the ability to eliminate the hyperplastic synovium by adenoviral-mediated gene transfer of human TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF family that is able to induce apoptosis through interaction with receptors containing death domains, DR4 and DR5. Infection of synovial cells derived from RA patients with Ad.TRAIL resulted in significant apoptosis in three out of five lines. Moreover, primary rabbit synovial fibroblasts were also sensitive to Ad.TRAIL-mediated gene transfer. In a rabbit model of arthritis, intra-articular gene transfer of TRAIL induced apoptosis in cells within the synovial lining, reduced leukocytic infiltration and stimulated new matrix synthesis by cartilage. These results demonstrate that TRAIL can affect the viability of the cells populating the activated synovium in arthritic joints and suggest that the delivery of TRAIL to arthritic joints may represent a non-invasive mechanism for inducing pannus regression.

Identification of fat-cell enhancer regions in Drosophila melanogaster.

The insect fat body is a dynamic tissue involved in maintaining homeostasis. It functions not only in energy storage and intermediary metabolism but also in detoxification, communication and the immune response. Some of these functions are confined to distinct groups of fat body cells. In Drosophila melanogaster, discrete precursor-cell clusters populate the fat body [Hoshizaki, D.K., Blackburn, T., Price, C., Ghosh, M., Miles, K., Ragucci, M. and Sweis, R. (1994) Embryonic fat-cell lineage in Drosophila melanogaster. Development 120: 2489-2499; Hoshizaki, D.K., Lunz, R., Ghosh, M. and Johnson, W. (1995) Identification of fat-cell enhancer activity in Drosophila melanogaster using P-element enhancer traps. Genome 38: 497-506; Riechmann, V., Rehorn, K.P., Reuter, R. and Leptin, M. (1998) The genetic control of the distinction between fat body and gonadal mesoderm in Drosophila. Development 125: 713-723]. Whether these clusters populate defined morphological regions or whether they represent the precursors to functionally similar groups of fat-body cells has not been formally demonstrated. We have identified a 2.1 kb enhancer region from serpent (srp), a GATA transcription factor gene that is sufficient to induce fat-cell formation. This enhancer region drives expression in specific groups of precursor-cell clusters, which we show give rise to defined regions of the mature embryonic fat body. We present evidence that srp expression in different precursor fat cells is controlled by independent cis-acting regulatory regions, and we have tested the role of trans-acting factors in the specification of some of these cells. We suggest that the different positional cues regulating srp expression, and therefore general fat-cell specification, might also be involved in the functional specialization of fat cells. This may be a common mechanism in insects to explain the origin of biochemically distinct regions of the larval/adult fat body.

Gene transfer of p53 to arthritic joints stimulates synovial apoptosis and inhibits inflammation.

Rheumatoid arthritis (RA) is an autoimmune disease that primarily affects joints. During the pathogenesis of rheumatoid arthritis, the synovial lining becomes dramatically thickened and hyperplastic. This highly aggressive tissue invades and destroys articular cartilage and bone. Several lines of evidence suggest that the proliferation of the synovial tissue may be due to disruption in the control of the cell cycle or apoptotic pathways. In particular, mutations in the tumor suppressor protein p53 have been found in synovial tissue from RA joints. We have examined the effects of overexpression of p53 by adenoviral infection in synovial cells in culture and in synovial tissue in vivo in a rabbit model of arthritis. Here we demonstrate that p53 overexpression resulted in significant apoptosis in human and rabbit synovial cells in culture. Furthermore, intraarticular injection of Ad-p53 resulted in extensive and rapid induction of synovial apoptosis in the rabbit knee without affecting cartilage metabolism. Interestingly, a significant reduction in the leukocytic infiltrate was observed within 24 h postinfection of Ad.p53. These results suggest that intraarticular gene transfer of p53 is able to induce synovial apoptosis as well as reduce inflammation and thus may be useful clinically for the treatment of RA.

Bcl-2 and GDNF delivered by HSV-mediated gene transfer act additively to protect dopaminergic neurons from 6-OHDA-induced degeneration.

Previous studies have demonstrated that either the neurotrophin glial-derived neurotrophic factor (GDNF) or the antiapoptotic peptide Bcl-2 delivered into striatum by a viral vector protects dopaminergic neurons of the substantia nigra in vivo from degeneration induced by the administration of the neurotoxin 6-hydroxydopamine (6-OHDA). In this study we used recombinant, replication-incompetent, genomic herpes simplex virus-based vectors to deliver the genes coding for Bcl-2 and GDNF into rat substantia nigra (SN) 1 week prior to 6-OHDA injection into the striatum. Vector-mediated expression of either Bcl-2 or GDNF alone each resulted in a doubling in cell survival as measured by retrograde labeling with fluorogold (FG) and a 50% increase in tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the lesioned SN compared to the unlesioned side. Gene transfer of Bcl-2 and GDNF were equivalent in this effect. Coadministration of the Bcl-2-expressing vector with the GDNF-expressing vector improved the survival of lesioned SN neurons as measured by FG labeling by 33% and by the expression of TH-IR by 15%. These results suggest that the two factors delivered together act in an additive fashion to improve DA cell survival in the face of 6-OHDA toxicity.

Future of adenoviruses in the gene therapy of arthritis.

Recombinant adenoviruses are straightforward to produce at high titres, have a promiscuous host-range, and, because of their ability to infect nondividing cells, lend themselves to in vivo gene delivery. Such advantages have led to their widespread and successful use in preclinical studies of arthritis gene therapy. While adenoviral vectors are well suited to 'proof of principle' experiments in laboratory animals, there are several barriers to their use in human studies at this time. Transient transgene expression limits their application to strategies, such as synovial ablation, which do not require extended periods of gene expression. Moreover, there are strong immunological barriers to repeat dosing. In addition, safety concerns predicate local, rather than systemic, delivery of the virus. Continued engineering of the adenoviral genome is producing vectors with improved properties, which may eventually overcome these issues. Promising avenues include the development of 'gutted' vectors encoding no endogenous viral genes and of adenovirus-AAV chimeras. Whether these will offer advantages over existing vectors, which may already provide safe, long-term gene expression following in vivo delivery, remains to be seen.

Herpes simplex virus vector-mediated expression of Bcl-2 protects spinal motor neurons from degeneration following root avulsion.

Proximal axotomy in adult animals results in delayed death of motor neurons. Features characteristic of both necrosis and apoptosis have been described in motor neurons of the spinal cord following proximal avulsion of the ventral roots. We have previously demonstrated that a genomic herpes simplex virus (HSV)-based vector expressing the anti-apoptotic peptide Bcl-2 protects dopaminergic neurons of the substantia nigra from neurotoxin-induced apoptotic cell death and preserves the neurotransmitter phenotype of those cells. In this study we examined whether the same vector could protect adult rat lumbar motor neurons from cell death following proximal ventral root avulsion. Injection of the Bcl-2-expressing vector 1 week prior to root avulsion increased the survival of lesioned motor neurons, determined by retrograde Fluorogold labeling, by 50%. The Bcl-2-expressing vector did not preserve choline acetyltransferase neurotransmitter phenotype of the lesioned cells. These results shed light on the mechanism of cell death following axonal injury, and have implications for developing an effective treatment for the clinical problem of proximal root avulsion.

Direct gene delivery strategies for the treatment of rheumatoid arthritis.

Gene therapy offers a novel and innovative approach to the delivery of therapeutic proteins to the joints of patients with arthritis. Several viral vectors, including adenovirus, adeno-associated virus, retrovirus and herpes simplex virus, are capable of delivering exogenous cDNAs to the synovial lining, enabling effective levels of intra-articular transgene expression following direct injection to the joint. The expression of certain gene products has proven to be sufficient to inhibit the progression of disease in animals with experimental arthritis. Non-viral methods of gene transfer, however, are less satisfactory, and are limited by toxicity and transience of expression. Although the principle of direct gene delivery to the joint has been demonstrated, maintaining persistent intra-articular transgene expression remains a challenge.

Effective treatment of established murine collagen-induced arthritis by systemic administration of dendritic cells genetically modified to express IL-4.

Dendritic cells (DC) are APCs that are able to stimulate or inhibit immune responses, depending on levels of expression of MHC class I and II costimulatory molecules and cytokines. Our previous studies have suggested that the observed contralateral effect, where injection of a vector carrying certain immunomodulatory genes into one joint resulted in inhibition of arthritis in untreated joints, is mediated by in vivo modification of DC. Therefore, we have examined the ability of genetically modified DC to suppress established murine collagen-induced arthritis (CIA) after i.v. delivery. IL-4 has been shown to partially reduce the severity of CIA after repeated injection of recombinant protein or by injection of an adenoviral vector expressing IL-4. Here we demonstrate that i.v. injection of immature DC, infected with an adenoviral vector expressing IL-4, into mice with established CIA resulted in almost complete suppression of disease, with no recurrence for up to 4 wk posttreatment. Injection i.v. of fluorescently labeled DC demonstrated that the cells rapidly migrated to the liver and spleen after 6 h and to the lymph nodes by 24 h. In culture, spleen cells from DC/IL-4-treated mice produced less IFN-gamma after stimulation by collagen than did control groups. In addition, DC/IL-4 administration decreased the level of specific Abs against type II collagen, in particular the IgG2 Th1 isotype 14 days posttreatment. These results demonstrate the ability to treat effectively established murine arthritis by systemic administration of DC expressing IL-4.

Gene therapy for established murine collagen-induced arthritis by local and systemic adenovirus-mediated delivery of interleukin-4.

To determine whether IL-4 is therapeutic in treating established experimental arthritis, a recombinant adenovirus carrying the gene that encodes murine IL-4 (Ad-mIL-4) was used for periarticular injection into the ankle joints into mice with established collagen-induced arthritis (CIA). Periarticular injection of Ad-mIL-4 resulted in a reduction in the severity of arthritis and joint swelling compared with saline- and adenoviral control groups. Local expression of IL-4 also reduced macroscopic signs of joint inflammation and bone erosion. Moreover, injection of Ad-mIL-4 into the hind ankle joints resulted in a decrease in disease severity in the untreated front paws. Systemic delivery of murine IL-4 by intravenous injection of Ad-mIL-4 resulted in a significant reduction in the severity of early-stage arthritis.

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.

Vector systems for gene transfer to joints.

The prospects for the development of gene therapy treatments for certain orthopaedic diseases have been fueled by advances in the understanding of the molecular components of these disorders. These studies have identified molecules that could have therapeutic or reparative effects in certain settings. The ability to transfer and appropriately express the genes encoding these molecules is dependent on the availability of effective gene transfer vectors. Numerous vector systems have been used to transfer and express genes in joints with varied levels of success. The current review is designed to briefly outline the basics of the different gene transfer vector systems available for use by researchers in the orthopaedic fields.

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.

Adenoviral mediated delivery of FAS ligand to arthritic joints causes extensive apoptosis in the synovial lining.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease where the synovial lining layer of the joint becomes thickened, hypercellular, and highly aggressive. Invading synovial tissue erodes cartilage and subchondral bone and leads to loss of joint function. FasL, a cell-surface molecule on activated T-cells interacts with its receptor, Fas, to induce apoptosis in target cells. We addressed the feasibility of using adenoviral gene transfer of FasL therapeutically to mediate apoptosis in arthritic joints similar in size to the small joints of the hands and feet that are the primary sites of RA in humans. METHODS: Adenoviral vectors were used to transfer FasL and LacZ cDNAs into human RA and rabbit synovial fibroblasts in culture where apoptosis was evaluated using MTT and TUNEL analyses. The ability of Ad.FasL to mediate synovial apoptosis in vivo was then addressed in an IL-1-induced arthritis model in the rabbit knee. RESULTS: In culture, delivery of FasL was found to efficiently induce apoptosis in both human RA and rabbit synovial fibroblasts. The ability of Ad.FasL to induce synovial apoptosis was then evaluated in rabbit knee joints. 24 h after intra-articular injection of 10(11) Ad.FasL particles, large regions of synovial tissue were observed histologically consisting primarily of fibrous matrix and cellular debris. TUNEL staining of corresponding sections was highly positive for fragmented DNA. Glycosaminoglycan (GAG) synthesis from cartilage shavings from treated joints suggests that Ad.FasL does not induce significant apoptosis in resident articular chondrocytes. CONCLUSIONS: Infection of human and rabbit synovial fibroblasts with Ad.FasL results in significant apoptotic cell death in vitro. Direct intra-articular injection of Ad.FasL in the arthritic rabbit knee results in extensive apoptosis in the synovium without affecting chondrocyte viability.

Cartilage injury and repair.

Articular cartilage is a complex structure that, once damaged, has little capacity for permanent repair. The problem lies in the inability of the body to regenerate tissue with the appropriate macromolecular constituents and architecture of normal hyaline cartilage. Although full-thickness defects are capable of stimulating a repair response, the resulting fibrocartilage is inferior and cannot withstand long-term, repetitive use. Numerous surgical approaches that involve penetration of subchondral bone offer short-term to moderate-term relief of symptoms, whereas other approaches have seen significant improvement through transplantation of osteochondral and periosteal tissue and implantation of autologous chondrocytes. Despite these procedures, articular cartilage damage continues to be an unmet clinical problem. Improvements in biochemical and molecular biologic techniques may allow advances in the understanding of chondrocyte and cartilage biology and may provide innovative and novel approaches to stimulating the repair of articular cartilage through biologic means.

Genetically modified CD34+ cells as cellular vehicles for gene delivery into areas of angiogenesis in a rhesus model.

To develop a cellular vehicle able to reach systemically disseminated areas of angiogenesis, we sought to exploit the natural tropism of circulating endothelial progenitor cells (EPCs). Primate CD34+ EPCs were genetically modified with high efficiency and minimal toxicity using a non-replicative herpes virus vector. These EPCs localized in a skin autograft model of angiogenesis in rhesus monkeys, and sustained the expression of a reporter gene for several weeks while circulating in the blood. In animals infused with autologous CD34+ EPCs transduced with a thymidine kinase-encoding herpes virus, skin autografts and subcutaneous Matrigel pellets impregnated with vascular growth factors underwent necrosis or accelerated regression after administration of ganciclovir. Importantly, the whole intervention was perfectly well tolerated. The accessibility, easy manipulation, lack of immunogenicity of the autologous CD34+ cell vehicles, and tropism for areas of angiogenesis render autologous CD34+ circulating endothelial progenitors as ideal candidates for exploration of their use as cellular vehicles when systemic gene delivery to those areas is required. Gene Therapy (2000) 7, 43-52.

Intra-articular delivery of a herpes simplex virus IL-1Ra gene vector reduces inflammation in a rabbit model of arthritis.

To evaluate the use of HSV-based vectors for arthritis gene therapy we have constructed a first-generation, ICP4 deficient, replication defective herpes simplex virus (HSV) vector (S/0-) and a second-generation HSV vector derivative (T/0-) deficient for the immediate-early genes ICP4, 22 and 27, each carrying a soluble TNF receptor or IL-1 receptor antagonist transgene cassette. A rabbit synovial-fibroblast line in culture, infected by either vector enabled high-level expression of the transgene product. However, following a single intra-articular injection of the vectors into rabbit knee joints, only the second-generation, HSV T/0- vector expressed detectable levels of soluble TNFR in synovial fluid. Synovial lavage fluid from inoculated joints con- tained up to 12 ng/ml of soluble receptor that persisted at detectable, but reduced levels for at least 7 days. When tested in an experimental model of arthritis generated by intra-articular overexpression of interleukin-1beta using retrovirus transduced synovial cells, the HSV T/0- vector expressing the interleukin-1 receptor antagonist was found to inhibit leukocytosis and synovitis significantly. The improved levels and duration of intra-articular transgene expression achieved via HSV-mediated gene delivery suggest that an HSV vector system could be used for therapeutic applications in patients with rheumatoid arthritis (RA) and other joint-related inflammatory diseases.

Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo.

6-Hydroxydopamine (6-OHDA) is widely used to selectively lesion dopaminergic neurons of the substantia nigra (SN) in the creation of animal models of Parkinson's disease. In vitro, the death of PC-12 cells caused by exposure to 6-OHDA occurs with characteristics consistent with an apoptotic mechanism of cell death. To test the hypothesis that apoptotic pathways are involved in the death of dopaminergic neurons of the SN caused by 6-OHDA, we created a replication-defective genomic herpes simplex virus-based vector containing the coding sequence for the antiapoptotic peptide Bcl-2 under the transcriptional control of the simian cytomegalovirus immediate early promoter. Transfection of primary cortical neurons in culture with the Bcl-2-producing vector protected those cells from naturally occurring cell death over 3 weeks. Injection of the Bcl-2-expressing vector into SN of rats 1 week before injection of 6-OHDA into the ipsilateral striatum increased the survival of neurons in the SN, detected either by retrograde labeling of those cells with fluorogold or by tyrosine hydroxylase immunocytochemistry, by 50%. These results, demonstrating that death of nigral neurons induced by 6-OHDA lesioning may be blocked by the expression of Bcl-2, are consistent with the notion that cell death in this model system is at least in part apoptotic in nature and suggest that a Bcl-2-expressing vector may have therapeutic potential in the treatment of Parkinson's disease.

Efficient gene delivery into epstein-barr virus (EBV)-transformed human B cells mediated by replication-defective herpes simplex virus-1 (HSV-1): A gene therapy model for EBV-related B cell malignancy.

Subgroups of the B cell malignancies are known to be associated with Epstein-Barr virus (EBV) infection, especially in immunocompromised patients. These are fatal and refractory to conventional antineoplastic therapy. B cells are usually post-mitotic cells and even mitogen activated or transformed B cells have shown relative resistance against viral mediated gene transfer. To address this issue, we employed a replication-defective herpes simplex virus-1 (HSV-1) to mediate gene transfer into EBV-transformed B cells. The virus expresses the herpes simplex virus thymidine kinase (HSV-TK) and the E. coli lacZ reporter genes and is designated T0Z.1. We used the lymphoblastoid cell line SWEIG as a model for human EBV-related B cell malignancy. This cell line was established by in vitro EBV infection of primary human peripheral blood mononuclear cells. When SWEIG cells were infected with T0Z.1, X-gal staining revealed lacZ expression in more than 20% cells even at multiplicity of infection (MOI) as low as 1 and the expression persisted for at least one week. Ganciclovir (GCV) administration after T0Z.1 infection effectively decreased the number of the infected tumor cells in a dose-responsive manner. Viral toxicity was analyzed by cell proliferation assay (MTS assay) and found to be little even at 10 MOI infection. Three MOI of the virus yielded maximum antineoplastic effect and more than 50% tumor cells were killed by HSV-TK/GCV. These results suggest the potential utility of replication-defective HSV-1 for the treatment of EBV-related B cell malignancies.