Age-dependent changes in intervertebral disc cell mitochondria and bioenergetics.

Robust cellular bioenergetics is vital in the energy-demanding process of maintaining matrix homeostasis in the intervertebral disc. Age-related decline in disc cellular bioenergetics is hypothesised to contribute to the matrix homeostatic perturbation observed in intervertebral disc degeneration. The present study aimed to measure how ageing impacted disc cell mitochondria and bioenergetics. Age-related changes measured included matrix content and cellularity in disc tissue, as well as matrix synthesis, cell proliferation and senescence markers in cell cultures derived from annulus fibrosus (AF) and nucleus pulposus (NP) isolated from the discs of young (6-9 months) and older (36-50 months) New Zealand White rabbits. Cellular bioenergetic parameters were measured using a Seahorse XFe96 Analyzer, in addition to quantitating mitochondrial morphological changes and membrane potential. Ageing reduced mitochondrial number and membrane potential in both cell types. Also, it significantly reduced glycolytic capacity, mitochondrial reserve capacity, maximum aerobic capacity and non-glucose-dependent respiration in NP. Moreover, NP cells exhibited age-related decline in matrix synthesis and reduced cellularity in older tissues. Despite a lack of changes in mitochondrial respiration with age, AF cells showed an increase in glycolysis and altered matrix production. While previous studies report age-related matrix degenerative changes in disc cells, the present study revealed, for the first time, that ageing affected mitochondrial number and function, particularly in NP cells. Consequently, age-related bioenergetic changes may contribute to the functional alterations in aged NP cells that underlie disc degeneration.

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.

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.

Orthopedic gene therapy--lost in translation?

Orthopedic gene therapy has been the topic of considerable research for two decades. The preclinical data are impressive and many orthopedic conditions are well suited to genetic therapies. But there have been few clinical trials and no FDA-approved product exists. This paper examines why this is so. The reasons are multifactorial. Clinical translation is expensive and difficult to fund by traditional academic routes. Because gene therapy is viewed as unsafe and risky, it does not attract major funding from the pharmaceutical industry. Start-up companies are burdened by the complex intellectual property environment and difficulties in dealing with the technology transfer offices of major universities. Successful translation requires close interactions between scientists, clinicians and experts in regulatory and compliance issues. It is difficult to create such a favorable translational environment. Other promising fields of biological therapy have contemplated similar frustrations approximately 20 years after their founding, so there seem to be more general constraints on translation that are difficult to define. Gene therapy has noted some major clinical successes in recent years, and a sense of optimism is returning to the field. We hope that orthopedic applications will benefit collaterally from this upswing and move expeditiously into advanced clinical trials.

Negative regulation of the Apaf-1 apoptosome by Hsp70.

Release of cytochrome c from mitochondria by apoptotic signals induces ATP/dATP-dependent formation of the oligomeric Apaf-1-caspase-9 apoptosome. Here we show that the documented anti-apoptotic effect of the principal heat-shock protein, Hsp70, is mediated through its direct association with the caspase-recruitment domain (CARD) of Apaf-1 and through inhibition of apoptosome formation. The interaction between Hsp70 and Apaf-1 prevents oligomerization of Apaf-1 and association of Apaf-1 with procaspase-9. On the basis of these results, we propose that resistance to apoptosis exhibited by stressed cells and some tumours, which constitutively express high levels of Hsp70, may be due in part to modulation of Apaf-1 function by Hsp70.

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.

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.

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.

Viral interleukin 10 (IL-10), the human herpes virus 4 cellular IL-10 homologue, induces local anergy to allogeneic and syngeneic tumors.

After the cloning of murine cytokine synthesis inhibitory factor, it was recognized that a homologous open reading frame was encoded within the Epstein-Barr virus (human herpes virus 4). This viral protein has now been termed viral interleukin 10 (vIL-10) to reflect its protein sequence homology to "cellular" IL-10 (cIL-10, either murine or human IL-10). It is now widely accepted that vIL-10 shares many functions with cIL-10, principally, the ability to enhance survival of newly infected B cells and to diminish the production of IFN-gamma and IL-2 during ongoing immune reactions. The immunomodulatory effect of locally secreted vIL-10 and murine IL-10 (mIL-10) was examined in tumor models using CL8-1 (a BL6 melanoma cell line transfected with the H-2Kb class I gene) in syngeneic animals. Although parental BL6 tumor cells grow in immunocompetent syngeneic hosts, CL8-1 are rejected. To achieve local secretion of vIL-10, we generated vIL-10 retroviral vectors. While nontransduced CL8-1 cells (1 x 10(4)) failed to grow when injected intradermally in C57BL/6 mice, CL8-1 cells (1 x 10(4)) transduced with vIL-10 formed palpable tumors and eventually killed 80% of injected animals. Suppression of tumor rejection was also noted when CL8-1 tumors with or without vIL-10 transfection were admixed with syngeneic vIL-10-transfected fibroblasts and inoculated. Since the in vitro proliferation of the tumor was not altered after transduction with the vIL-10 gene and injection of vIL-10-transduced CL8-1 does not affect the rejection of nontransduced CL8-1 inoculated at a distant site, local vIL-10 secretion appears to suppress the process of immune rejection of the target cells in a dose-dependent manner. Similar results were observed for the H-2b MCA105 sarcoma tumor model in allogeneic BALB/c mice (H-2d). Although all animals that received nontransfected MCA105 rapidly rejected these tumors, MCA105 sarcomas transfected with vIL-10 remained palpable for up to 37 d. The local immunosuppressive effect of gene-delivered vIL-10 could be neutralized by anti-human IL-10 monoclonal antibody or could be reversed by the systemic administration of IL-2 or IL-12. In marked contrast, mIL-10 transfection of CL8-1 significantly suppressed tumor growth and frequently led to the rejection of tumor. Similar results were obtained for the murine tumor cell lines MCA102.(ABSTRACT TRUNCATED AT 400 WORDS)

Progress and Prospects: genetic treatments for disorders of bones and joints.

Gene therapies directed toward the treatment of arthritis and tissue repair continue to be the most active areas of research for bone and joint diseases. In the past 2 years, two trials in rheumatoid arthritis have been completed. a Phase I study reporting safety and a Phase I/II study that has yet to be published. An additional, small study has reported the first evidence of clinical efficacy. Two Phase I trials of gene therapy for osteoarthritis have also been initiated. There is much preclinical activity in developing AAV vectors for future trials in the gene therapy of arthritis. Research into tissue repair and regeneration remains at the preclinical stage, but a considerable volume of research attests to the promise of gene transfer in this arena, especially in the context of bone healing. For tissue repair, the major research questions are still which genes to use and how best to deliver them.

Development of approaches to improve cell survival in myoblast transfer therapy.

Myoblast transplantation has been extensively studied as a gene complementation approach for genetic diseases such as Duchenne Muscular Dystrophy. This approach has been found capable of delivering dystrophin, the product missing in Duchenne Muscular Dystrophy muscle, and leading to an increase of strength in the dystrophic muscle. This approach, however, has been hindered by numerous limitations, including immunological problems, and low spread and poor survival of the injected myoblasts. We have investigated whether antiinflammatory treatment and use of different populations of skeletal muscle-derived cells may circumvent the poor survival of the injected myoblasts after implantation. We have observed that different populations of muscle-derived cells can be isolated from skeletal muscle based on their desmin immunoreactivity and differentiation capacity. Moreover, these cells acted differently when injected into muscle: 95% of the injected cells in some populations died within 48 h, while others richer in desmin-positive cells survived entirely. Since pure myoblasts obtained from isolated myofibers and myoblast cell lines also displayed a poor survival rate of the injected cells, we have concluded that the differential survival of the populations of muscle-derived cells is not only attributable to their content in desmin-positive cells. We have observed that the origin of the myogenic cells may influence their survival in the injected muscle. Finally, we have observed that myoblasts genetically engineered to express an inhibitor of the inflammatory cytokine, IL-1, can improve the survival rate of the injected myoblasts. Our results suggest that selection of specific muscle-derived cell populations or the control of inflammation can be used as an approach to improve cell survival after both myoblast transplantation and the myoblast-mediated ex vivo gene transfer approach.

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.

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.

Tolerogenic dendritic cells for autoimmune disease and transplantation.

Dendritic leucocytes are professional antigen-presenting cells with inherent tolerogenic properties and are regarded as critical regulators of innate and adaptive immunity. Modification of dendritic cells (DCs) in the laboratory can enhance and stabilise their tolerogenic properties. Numerous reports suggest that such immature, maturation-resistant or "alternatively activated" DCs can regulate autoreactive or alloreactive T-cell responses and promote or restore antigen-specific tolerance in experimental animal models. The first clinical testing of tolerogenic DCs in human autoimmune disease, including rheumatoid arthritis, is imminent. Herein the properties of tolerogenic DCs and prospects for their testing in chronic inflammatory disease and transplantation are reviewed.

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.

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.

Rb inhibits the intrinsic kinase activity of TATA-binding protein-associated factor TAFII250.

The retinoblastoma tumor suppressor protein, Rb, interacts directly with the largest TATA-binding protein-associated factor, TAFII250, through multiple regions in each protein. To define the potential role(s) of this interaction, we examined whether Rb could regulate the intrinsic, bipartite kinase activity of TAFII250. Here, we report that Rb is able to inhibit the kinase activity of immunopurified and gel-purified recombinant TAFII250. Rb inhibits the autophosphorylation of TAFII250 as well as its phosphorylation of the RAP74 subunit of TFIIF in a dose-responsive manner. Inhibition of TAFII250 kinase activity involves the Rb pocket (amino acids 379 to 928) but not its amino terminus. In addition, Rb appears to specifically inhibit the amino-terminal kinase domain of TAFII250 through a direct protein-protein interaction. We further demonstrate that two different tumor-derived Rb pocket mutants, C706F and Deltaex22, are functionally defective for kinase inhibition, even though they are able to bind the amino terminus of TAFII250. Our results suggest a novel mechanism of transcriptional regulation by Rb, involving direct interaction with TAFII250 and inhibition of its ability to phosphorylate itself, RAP74, and possibly other targets.

trans Activation of the simian virus 40 enhancer.

We describe experiments which demonstrated that the simian virus 40 (SV40) enhancer affects certain transcriptional units differently. We also found that a specific enhancer-transcriptional unit interaction can be regulated by trans-acting factors. Using transient assays, we examined the effects of the SV40 enhancer on herpesvirus thymidine kinase (tk) RNA levels when transcription was initiated either by the herpesvirus tk promoter or by an SV40 early promoter-tk fusion. We were unable to detect any effect of the enhancer on transcription from the tk promoter in CV-1 or HeLa cells. However, we found that the addition of T-antigen in trans allowed the enhancer to stimulate expression from the tk promoter. This induction by T-antigen did not require T-antigen-binding sites in cis and appeared to be an indirect effect. In contrast, tk expression from the SV40 early promoter fusion was greatly stimulated by the enhancer in CV-1 cells. Furthermore, in 293 cells the SV40 enhancer had only a marginal effect on the SV40 promoter-tk fusion, whereas it strongly stimulated tk expression from the tk promoter. Our results raise the possibility that the enhancer function may not show cell specificity per se; rather, the interaction between the enhancer and a specific gene may be responsible for cell specificity. We discuss these observations in terms of the SV40 early gene-to-late gene switch that occurs during SV40 lytic growth.

The retinoblastoma gene product regulates Sp1-mediated transcription.

We have demonstrated that the retinoblastoma gene product (Rb) can positively regulate transcription from the fourth promoter of the insulinlike growth factor II gene. Two copies of a motif (the retinoblastoma control element) similar to that found in the human c-fos, transforming growth factor beta 1, and c-myc promoters are responsible for conferring Rb regulation to the fourth promoter of the insulinlike growth factor II gene. We have shown that the transcription factor Sp1 can bind to and stimulate transcription from the retinoblastoma control element motif. Moreover, by using a GAL4-Sp1 fusion protein, we have directly demonstrated that Rb positively regulates Sp1 transcriptional activity in vivo. These results indicate that Rb can function as a positive regulator of transcription and that Sp1 is one potential target, either directly or indirectly, for transcriptional regulation by Rb.