NF-κB inhibition reveals a novel role for HGF during skeletal muscle repair.

The transcription factor nuclear factor κB (NF-κB)/p65 is the master regulator of inflammation in Duchenne muscular dystrophy (DMD). Disease severity is reduced by NF-κB inhibition in the mdx mouse, a murine DMD model; however, therapeutic targeting of NF-κB remains problematic for patients because of its fundamental role in immunity. In this investigation, we found that the therapeutic effect of NF-κB blockade requires hepatocyte growth factor (HGF) production by myogenic cells. We found that deleting one allele of the NF-κB subunit p65 (p65+/-) improved the survival and enhanced the anti-inflammatory capacity of muscle-derived stem cells (MDSCs) following intramuscular transplantation. Factors secreted from p65+/- MDSCs in cell cultures modulated macrophage cytokine expression in an HGF-receptor-dependent manner. Indeed, we found that following genetic or pharmacologic inhibition of basal NF-κB/p65 activity, HGF gene transcription was induced in MDSCs. We investigated the role of HGF in anti-NF-κB therapy in vivo using mdx;p65+/- mice, and found that accelerated regeneration coincided with HGF upregulation in the skeletal muscle. This anti-NF-κB-mediated dystrophic phenotype was reversed by blocking de novo HGF production by myogenic cells following disease onset. HGF silencing resulted in increased inflammation and extensive necrosis of the diaphragm muscle. Proteolytic processing of matrix-associated HGF is known to activate muscle stem cells at the earliest stages of repair, but our results indicate that the production of a second pool of HGF by myogenic cells, negatively regulated by NF-κB/p65, is crucial for inflammation resolution and the completion of repair in dystrophic skeletal muscle. Our findings warrant further investigation into the potential of HGF mimetics for the treatment of DMD.

Renal cell carcinoma to haemangioblastoma metastasis: a rare manifestation of Von Hippel-Lindau syndrome.

Brain metastases are the most common intracranial malignancy in adults and may occasionally deposit within a pre-existing primary brain neoplasm. We describe, in two directly related family members, the rare occurrence of renal cell carcinoma (RCC) metastasis to haemangioblastoma (HB) in the context of Von Hippel-Lindau syndrome. Detection of this phenomenon can be marred by histological overlap between RCC and HB and therefore careful histological examination, and consideration of supportive immunohistochemistry, is required when examining all HB resections. Metastatic RCC to HB upstages a primary RCC and is clinically diagnostic of Von Hippel-Lindau syndrome.

Expression of IL-2 in ß cells by AAV8 gene transfer in pre-diabetic NOD mice prevents diabetes through activation of FoxP3-positive regulatory T cells.

We previously demonstrated that intraperitoneal delivery of adeno-associated virus serotype 8 (AAV8) stably transduces the pancreas, including the ß cells in the pancreatic islets. We further demonstrated the ability to deliver and express target genes specifically in ß cells for at least 6 months using a murine insulin promoter in a double-stranded, self-complementary AAV vector. Recombinant interleukin (IL)-2 has been shown to induce CD4(+)CD25(+) regulatory T cells (Tregs) in several mouse models of autoimmune disease. Here we evaluated the effects of double-stranded adeno-associated virus serotype 8-mouse insulin promoter (dsAAV8-mIP)-mediated delivery of  2 to pancreatic ß cells in non-obese diabetic (NOD) mice. AAV8-mIP-mediated gene expression of IL-2 to pancreatic ß cells of 10-week-old NOD mice prevented the onset of hyperglycemia in NOD mice more in a dose-dependent manner with the lower dose of virus being more effective than a higher dose of AAV-mIP-IL-2 and IL-4. Moreover, the local ß-cell expression of IL-2 increased the number of CD4(+)CD25(+)FoxP3(+) cells in the pancreatic lymph node (PLN) and SPL in both NOD and C57BL/6 mice. Taken together, these results demonstrate that local, low expression of mIL-2 in islets prevents progress of diabetes through the regulation of Tregs.

Exosomes in the Pathogenesis, Diagnosis and Treatment of Pancreatic Diseases.

Exosomes (EXOs) are small vesicles (30-200 nm) of endocytic origin, which are released by many different cell types into the extracellular space. They may play a key role in facilitating cell-cell communication, under both physiological and pathological conditions. EXOs contain a wide range of RNA molecules and proteins. Their specific molecular signatures make them promising candidates in early diagnosis and prognosis of pancreatic diseases. EXOs could also provide a new method to monitor treatment response in patients suffering from pancreatic cancer and other diseases of the pancreas. Additionally they may help to improve current treatments via personalized medicine approaches using them as therapeutic vehicles themselves.

LIM domain protein-3 (LMP3) cooperates with BMP7 to promote tissue regeneration by ligament progenitor cells.

Gene transfer of key regulators of osteogenesis for mesenchymal stem cells represents a promising strategy to regenerate bone. It has been reported that LMP3, a transcription variant of LIM domain mineralization protein (LMP) lacking LIM domains, can induce osteogenesis in vitro and in vivo. As little is known about the effects of LMP3 gene therapy on periodontal ligament (PDL) cell osteogenic differentiation, this study sought to explore whether gene delivery of LMP3 can promote PDL cell mineralization and bone formation. Our results showed that adenoviral mediated gene transfer of LMP3 (AdLMP3) significantly upregulated ALP (Alkaline Phosphatase), BSP (Bone Sialoprotein) and BMP2 gene expression and increased in vitro matrix mineralization in human PDL. Although AdLMP3 gene delivery to PDL cells did not induce ectopic bone formation in vivo, we found that AdLMP3 augments new bone formation, which co-delivered with AdBMP7 gene transfer. Our study provides the evidence that there is a synergistic effect between LMP3 and BMP-7 in vivo, suggesting that LMP3 delivery may be used to augment BMP-mediated osteogenesis. LMP3 and BMP-7 combinatory gene therapy may also have specific applications for oral and periodontal regenerative medicine.

dsAAV8-mediated gene transfer and ß-cell expression of IL-4 and ß-cell growth factors are capable of reversing early-onset diabetes in NOD mice.

Type-I diabetes is a chronic disease mediated by autoimmune destruction of insulin-producing ß-cells. Although progress has been made towards improving diabetes-associated pathologies and the quality of life for those living with diabetes, no therapy has been effective at eliminating disease manifestations or reversing disease progression. Here, we examined whether double-stranded adeno-associated virus serotype 8 (dsAAV8)-mediated gene delivery to endogenous ß-cells of interleukin (IL)-4 in combination with ß-cell growth factors can reverse early-onset diabetes in NOD mice. Our results demonstrate that a single treatment with dsAAV8 vectors expressing IL-4 in combination with glucagon-like peptide-1 or hepatocyte growth factor/NK1 under the regulation of the insulin promoter enhanced ß-cell proliferation and survival in vivo, significantly delaying diabetes progression in NOD mice, and reversing disease in ∼10% of treated NOD mice. These results demonstrate the ability to reverse hyperglycemia in NOD mice with established diabetes by in vivo gene transfer to ß-cells of immunomodulatory factors and ß-cell growth factors.

The antitumor effects of adenoviral-mediated, intratumoral delivery of interleukin 23 require endogenous IL-12.

Interleukin (IL)-23 is a member of the IL-12 family of heterodimeric cytokines, comprised of p19 and p40 subunits, which exhibits immunostimulatory properties similar to IL-12. We have demonstrated previously that adenoviral-mediated, intratumoral delivery of IL-23 (Ad.IL-23) was able to induce systemic antitumor immunity. Here we demonstrate that Ad.IL-23 requires endogenous IL-12 for conferring an antitumor effect after adenoviral-mediated, intratumoral delivery. In contrast, Ad.IL-12 does not require IL-23 for its antitumor effects although endogenous IL-23 appears important for induction of systemic antitumor immunity by IL-12. However, despite the requirement for endogenous IL-12, co-delivery of IL-23 and IL-12 does not provide even an additive local or systemic antitumor effect, regardless of the dose. We further demonstrate that although the use of a single-chain IL-23 (scIL-23) results in higher level of expression and a more pronounced IL-23-mediated antitumor effect, there is still no synergy with IL-12. These results demonstrate that although significant antitumor effects are achieved by intratumoral injection of adenovirus expressing either scIL-23 or IL-12 alone and that IL-23 requires endogenous IL-12 for maximum antitumor benefit, the combined use of these cytokines provides no additive or synergistic effect.

Bioplasty for vertebral fractures: preliminary results of a pre-clinical study on goats using autologous modified skin fibroblasts.

The debate is still ongoing about the long term effects of the mininvasive vertebral augmentation techniques and their usefulness in treating more complex cases where a bone inducing effect more than a merely bone substitution would be suitable, such as the vertebral fractures in young patients. We previously developed a clinically relevant gene therapy approach using modified dermal fibroblasts for inducing bone healing and bone formation in different animal models. The aim of this study is to show the feasibility of a minimally invasive percutaneous intrasomatic ex vivo gene therapy approach to treat thoracolumbar vertebral fractures and anterior column bone defects in a goat model.

Inhibition of the IKK/NF-κB pathway by AAV gene transfer improves muscle regeneration in older mdx mice.

The IκB kinase (IKKα, ß and the regulatory subunit IKKγ) complex regulates nuclear factor of κB (NF-κB) transcriptional activity, which is upregulated in many chronic inflammatory diseases. NF-κB signaling promotes inflammation and limits muscle regeneration in Duchenne muscular dystrophy (DMD), resulting in fibrotic and fatty tissue replacement of muscle that exacerbates the wasting process in dystrophic muscles. Here, we examined whether dominant-negative forms of IKKα (IKKα-dn) and IKKß (IKKß-dn) delivered by adeno-associated viral (AAV) vectors to the gastrocnemius (GAS) and tibialis anterior (TA) muscles of 1, 2 and 11-month-old mdx mice, a murine DMD model, block NF-κB activation and increase muscle regeneration. At 1 month post-treatment, the levels of nuclear NF-κB in locally treated muscle were decreased by gene transfer with either AAV-CMV-IKKα-dn or AAV-CMV-IKKß-dn, but not by IKK wild-type controls (IKKα and ß) or phosphate-buffered saline (PBS). Although treatment with AAV-IKKα-dn or AAV-IKKß-dn vectors had no significant effect on muscle regeneration in young mdx mice treated at 1 and 2 months of age and collected 1 month later, treatment of old (11 months) mdx with AAV-CMV-IKKα-dn or AAV-CMV-IKKß-dn significantly increased levels of muscle regeneration. In addition, there was a significant decrease in myofiber necrosis in the AAV-IKKα-dn- and AAV-IKKß-dn-treated mdx muscle in both young and old mice. These results demonstrate that inhibition of IKKα or IKKß in dystrophic muscle reduces the adverse effects of NF-κB signaling, resulting in a therapeutic effect. Moreover, these results clearly demonstrate the therapeutic benefits of inhibiting NF-κB activation by AAV gene transfer in dystrophic muscle to promote regeneration, particularly in older mdx mice, and block necrosis.

DsAAV8-mediated expression of glucagon-like peptide-1 in pancreatic beta-cells ameliorates streptozotocin-induced diabetes.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that performs a wide array of well-characterized antidiabetic actions, including stimulation of glucose-dependent insulin secretion, upregulation of insulin gene expression and improvements in beta-cell survival. GLP-1-receptor agonists have been developed for treatment of diabetes; however, the short biological half-lives of these peptide-based therapeutics requires that frequent injections be administered to maintain sufficient circulating levels. Thus, novel methods of delivering GLP-1 remain an important avenue of active research. It has recently been demonstrated that self-complimentary, double-stranded, adeno-associated virus serotype-8 (DsAAV8) can efficiently transduce pancreatic beta-cells in vivo, resulting in long-term transgene expression. In this study, we engineered a DsAAV8 vector containing a GLP-1 transgene driven by the mouse insulin-II promoter (MIP). Biological activity of the GLP-1 produced from this transgene was assessed using a luciferase-based bioassay. DsAAV8-MIP-GLP-1 was delivered via intraperitoneal injection and beta-cell damage induced by multiple low dose streptozotocin (STZ) administration. Glucose tolerance was assessed following intraperitoneal glucose injections and beta-cell proliferation measured by PCNA expression. Expression of GLP-1 in Min6 beta-cells resulted in glucose-dependent secretion of biologically active GLP-1. Intraperitoneal delivery of DsAAV8-MIP-GLP-1 to mice led to localized GLP-1 expression in beta-cells and protection against development of diabetes induced by multiple low-dose STZ administration. This protection was associated with significant increase in beta-cell proliferation. Results from this study indicate that expression and secretion of GLP-1 from beta-cells in vivo via DsAAV8 represents a novel therapeutic strategy for treatment of diabetes.

Adenoviral-mediated, intratumor gene transfer of interleukin 23 induces a therapeutic antitumor response.

Interleukin 23 (IL-23) is a member of the IL-12 family of heterodimeric cytokines, composed of p19 and p40 subunits, which exhibits immunostimulatory properties similar to IL-12. IL-23 has been shown to possess potent antitumor activities in several establishment models of cancer and a few therapeutic models, but the efficacy of local, adenoviral-mediated expression of IL-23 in established tumors has yet to be investigated. Here we have examined the antitumor activity of adenovirally delivered IL-23 in a day-7 MCA205 murine fibrosarcoma tumor model. Three intratumoral injections of adenovirus expressing IL-23 (Ad.IL-23) significantly increased animal survival and resulted in complete rejection of 40% of tumors, with subsequent generation of protective immunity and MCA205-specific cytotoxic T lymphocytes. In addition, we have shown that the antitumor activity of IL-23 is independent of IL-17, perforin and Fas ligand, but dependent on interferon-gamma, CD4(+) and CD8(+) T cells. These results demonstrate that direct intratumoral injection of adenovirus expressing IL-23 results in enhanced survival, tumor eradication and generation of protective immunity by generation of a Th1-type immune response.

Intravascular lymphoma presenting as progressive paraparesis.

We present a patient with subacute progressive paraparesis secondary to intravascular lymphoma restricted to the spinal cord where initial laboratory and imaging studies were inconclusive. We emphasise the importance of a systematic approach to the diagnosis and highlight the utility of spinal cord biopsy to establish the definitive diagnosis of this rare but treatable illness.

Ex vivo-transduced autologous skin fibroblasts expressing human Lim mineralization protein-3 efficiently form new bone in animal models.

Local gene transfer of the human Lim mineralization protein (LMP), a novel intracellular positive regulator of the osteoblast differentiation program, can induce efficient bone formation in rodents. To develop a clinically relevant gene therapy approach to facilitate bone healing, we have used primary dermal fibroblasts transduced ex vivo with Ad.LMP-3 and seeded on a hydroxyapatite/collagen matrix prior to autologous implantation. Here, we demonstrate that genetically modified autologous dermal fibroblasts expressing Ad.LMP-3 are able to induce ectopic bone formation following implantation of the matrix into mouse triceps and paravertebral muscles. Moreover, implantation of the Ad.LMP-3-modified dermal fibroblasts into a rat mandibular bone critical size defect model results in efficient healing, as determined by X-rays, histology and three-dimensional microcomputed tomography (3DmuCT). These results demonstrate the effectiveness of the non-secreted intracellular osteogenic factor LMP-3 in inducing bone formation in vivo. Moreover, the utilization of autologous dermal fibroblasts implanted on a biomaterial represents a promising approach for possible future clinical applications aimed at inducing new bone formation.

Small molecules in stem cell self-renewal and differentiation.

The use of stem cells in regenerative medicine is a promising approach to the treatment of disease and injury. Natural and synthetic small molecules have been shown to be useful chemical tools for controlling and manipulating the fates of cells. Small molecules can target signaling transduction pathways (for example, tyrosine kinase receptors) and affect DNA replication, cell differentiation, tumor metastasis and apoptosis. Stem cells share many properties with cancer cells and these similarities can provide insights to control and direct cell behavior; small molecules are already standard chemotherapeutics in the treatment of cancer. Libraries of small molecules have been examined for anticancer behavior (especially apoptosis), and, more recently, for stem cell self-renewal and differentiation capabilities in potential approaches to regenerative medicine. Differentiation therapy for cancer is based on the idea that cancer cells are undifferentiated embryonic-like cells and proposes to promote the differentiation and hence block cell proliferation. For example, retinoids have a role in stem cell differentiation to several lineages and have also been used to promote differentiation of acute promyeloic leukemic cells. Small molecules are also important tools for understanding mechanistic and developmental processes. Strategies for generating functional small molecule libraries have been outlined previously. In this review, we will look at several small molecules that have been described in the recent literature as effectors of stem cell self-renewal or differentiation as associated with the Wnt, Hedgehog or NF-kappaB pathways.

Protein transduction: identification, characterization and optimization.

Protein transduction domains (PTDs), both naturally occurring and synthetic, have been increasingly employed to deliver biologically active agents to a variety of cell types in vitro and in vivo. In addition to the previously characterized arginine-rich PTDs, including Tat (transactivator of transcription), Antp (Antennapedia) and PTD-5, we have demonstrated that lysine and ornithine, as well as arginine, homopolymers are able to mediate transduction of a wide variety of agents. To screen for optimal PTDs, we have used as a therapeutic cargo a peptide derived from IKK {IkappaB [inhibitor of NF-kappaB (nuclear factor kappaB)] kinase} beta, able to bind to the IKK regulatory subunit [NEMO (NF-kappaB essential modulator)], preventing formation of an active kinase complex. This peptide, termed NBD, is able to block activation of NF-kappaB, but not basal activity. We demonstrate that PTD-mediated delivery of NBD using certain PTDs, in particular 8K (octalysine), is therapeutic following systemic delivery in murine models of inflammatory bowel disease, diabetes and muscular dystrophy. In addition, we have developed a peptide phage display library screening method for novel transduction peptides able to facilitate tissue-specific internalization of marker protein complexes. Using this approach, we have identified transduction peptides that are able to facilitate internalization of large protein complexes into tumours, airway epithelia, synovial fibroblasts, cardiac tissue and HEK-293 (human embryonic kidney) cells in culture and/or in vivo.

Genetic modification of chondrocytes with insulin-like growth factor-1 enhances cartilage healing in an equine model.

Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 x 10(7) AdIGF-1 modified chondrocytes and the contralateral joint received 2 x 10(7) naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated. Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and correlated with increased collagen type II immunoreaction up to eight months. Genetic modification of chondrocytes with AdIGF-1 prior to transplantation improved early (four to nine weeks), and to a lesser degree long-term, cartilage healing in the equine model. The equine model of cartilage healing closely resembles human clinical cartilage repair. The results of this study suggest that cartilage healing can be enhanced through genetic modification of chondrocytes prior to transplantation.

Targeting of products of genes to tumor sites using adoptively transferred A-NK and T-LAK cells.

Despite successes in animals, cytokine gene expression selectively in human tumors is difficult to achieve owing to lack of efficient delivery methods. Since interleukin (IL)-2-activated natural killer (A-NK) and phytohemagglutinin and IL-2 activated killer T (T-LAK) cells, as previously demonstrated, localize and accumulate in murine lung tumor metastases following adoptive transfer, we transduced them to test their ability to deliver products of genes selectively to tumors. Assessments of transduction efficiency in vitro demonstrated that adenoviral transduction consistently resulted in high (>60%) transduction rates and substantial expression of transgenes such as GFP, Red2, luciferase, beta-galactosidase and mIL-12 for at least 4 days. In vivo experiments illustrated that Ad-GFP transduced A-NK and Ad-Red2 (RFP) transduced T-LAK or mIL-12 transduced A-NK cells localized 10-50-fold more or survived significantly better than mock transduced cells, respectively, within lung metastases than in the surrounding normal lung tissue. Most importantly, mIL-12 transduced A-NK cells provided a significantly greater antitumor response than non-transduced A-NK cells. Thus, adoptive transfer of A-NK and T-LAK cells represents an efficient method for targeting products of genes to tumor sites.

Direct adenovirus-mediated IGF-I gene transduction of synovium induces persisting synovial fluid IGF-I ligand elevations.

Insulin-like growth factor-I (IGF-I) is one of the most influential growth factors in cartilage repair. Maintenance of adequate IGF-I levels after articular repair procedures is complicated by the short biological half-life of IGF-I in vivo. This study investigated the potential for more prolonged IGF-I delivery through direct adenoviral mediated transduction of synovial tissues in the metacarpophalangeal (MCP) joints of horses. The use of a large animal model provided a structurally similar and metabolically relevant corollary to the human knee. The complete IGF-I coding sequence was packaged into an E1-E3 deleted adenovirus-5 vector under cytomegalovirus promoter control (AdIGF-I), and injected at varying total joint doses to the MCP joints of 14 horses. Direct injection of 20 and 50 x 10(10) AdIGF-I resulted in significant elevations of IGF-I in synovial fluid for approximately 21 days. Synovial tissue taken from injected joints at day 35 following injection and compared to tissue taken preinjection from the same joints revealed elevated synoviocyte IGF-I mRNA levels for the highest viral dose by in situ hybridization and real-time PCR techniques. AdIGF-I injections did not result in significant lameness, joint effusion or elevated total protein concentrations in the synovial fluid. Mild mononuclear infiltration of white blood cells was evident in histologic sections of the synovium in the second highest adenoviral IGF-I dose of 20 x 10(10) particles. Cartilage biopsies taken from all injected joints did not reveal any significant changes in proteoglycan levels nor in histological morphology, which included chondrocyte cloning, architecture, cell type or toluidine blue staining, when compared to control joints. Based on these findings, gene transfer of IGF-I to the synovium of joints can result in significant and persistent elevations of IGF-I ligand in synovial fluid with minimal detrimental effects. Direct IGF-I gene therapy may offer a simple approach in treating patients with acute cartilage injury.

Effect of BMP-2 gene transfer on bone healing in sheep.

Critical size defects of bone and delayed fracture healing due to metabolic disorders are still problems in orthopaedic surgery. Adenoviral vectors encoding bone morphogenetic protein-2 (Ad.BMP-2) have been used to stimulate bone formation in small animals. The present study evaluated the use of direct adenoviral gene transfer for inducing bone formation in a large animal. Standardized iliac crest defects were created surgically on both sides of the pelvic bone of white mountain sheep. The efficiency of gene transfer was evaluated using recombinant adenoviruses carrying the cDNA for luciferase. High levels of transgene expression, restricted to the site of injection, were found for the 1st week. Transgene expression then fell considerably, but could still be detected for up to 5 weeks. To investigate the effect on bone healing, Ad.BMP-2 (10(11) particles in 200 mul saline) was unilaterally injected into iliac crest defects and into tibial osteotomies. The contralateral defects remained untreated to evaluate possible systemic effects. The controls were treated with saline solution. Bone formation within the defect, assessed by micro-computed tomography (CT) measurement at 8 weeks, and callus formation after osteotomy were significantly reduced following direct application of Ad.BMP-2. The retardation compared to untreated control animals was additionally found at the contralateral iliac crest indicating a systemic inhibitory effect. Histological analysis confirmed the CT measurement and showed an increased number of inflammatory cells within both defects. Antibodies against the adenovirus and the transgene product were detected in all treated animals. These data show a systemic retardation of bone formation following a single local injection of Ad.BMP-2 in sheep. This finding stands in contrast to the data obtained from small animal models. Further studies are needed to determine the contribution of the immune response to these results, and whether a lower dose of Ad.BMP-2 would be advantageous.

Gene therapeutic approaches-transfer in vivo.

Osteoarthritis (OA) is common, debilitating, expensive, incurable and very difficult to treat. Gene transfer to the synovial linings of affected joints is a promising strategy for achieving sustained, therapeutic, intraarticular concentrations of anti-arthritic gene products. This is not reasonably possible with existing, alternative technologies. The present review summarizes progress in achieving direct, in vivo intraarticular gene delivery and expression. Numerous non-viral vectors have been evaluated for their ability to transfect the synovia of experimental animals following intraarticular injection. None have given more than low levels of temporary transgene expression and many are inflammatory. Several viral vectors, however, are very effective in this regard and successfully treat experimental models of OA. Adeno-associated virus has been used in a phase I study for the gene therapy of rheumatoid arthritis. Its use in a clinical trial for treating OA is pending.