Paul D. Robbins, PhD

Gene Therapy for Arthritis: We have been developing gene therapy approaches for treating arthritis and other connective tissue diseases using in vivo and ex vivo methods for gene delivery to joints. In particular, we have used an ex vivo approach where synovial cells are isolated from the rabbit knee by partial synovectomy, propagated in culture, infected with a retroviral vector, and subsequently transplanted back into the rabbit knee. The intra-articular expression of IL-1Ra protein by genetically modified, transplanted synoviocytes dramatically reduced all pathophysiological parameters associated with arthritis in a rabbit knee model. These results, demonstrating the feasibility and efficacy of treating arthritis by gene transfer, have led to a recently completed Phase I clinical protocol to treat rheumatoid arthritis by gene therapy. We also have utilized replication-defective adenovirus vector to deliver genes encoding anti-inflammatory agents (vIL-10, IL-10, IL-4, IL-1Ra, IL-1 and TNF-alpha soluble receptors), growth factors, (IGF-1, BMP-2 and TGF-ß), and pro-apoptotic agents (p53, FasL, and TRAIL) to the knee joints of rabbits with antigen-induced arthritis (a.i.a.). Intra-articular expression of the inflammatory proteins significantly reduced leukocytosis, cartilage matrix degradation, inhibition of new cartilage synthesis as well as the degree of synovitis. Gene transfer of IGF-1 stimulated repair of damaged cartilage whereas gene transfer of the apoptotic agents induced apoptosis of the proliferating synovium. Interestingly, induction of synovial apoptosis resulted in a significant reduction in the inflammation. In addition to adenoviral vectors, we also have examined and compared the efficiency of a panel of viral and non-viral vectors including HSV, AAV and transposable elements for gene transfer to the rabbit knee. During the course of the experiments examining the efficacy of adenoviral mediated gene transfer of IL-1 and TNF inhibitory genes, an anti-arthritic effect was also observed in opposing contralateral control knee joints that received only a marker gene. Analysis of the contralateral effect has shown that it is antigen specific and is conferred, at least in part, by functionally altered dendritic cells as well as exosomes derived from dendritic cells. The recent focus of the laboratory has been on 1) development of direct delivery methods, using both viral and non-viral vectors, for transferring genes to rabbit synovium; 2) screening of potential therapeutic genes including IL-1 and TNF-alpha inhibitors, anti-inflammatory cytokines, soluble adhesion factors, and TIMPs; 3) development of methods for treating autoimmune diseases systemically through the delivery of therapeutic proteins using T-cells and dendritic cells; and 4) using gene transfer to examine the role of specific proteins in the pathogenesis of arthritis. 

Immunosuppressive DC and Exosomes: Dendritic cells (DC) are professional antigen presenting cells (APC) that are able to modulate T cell immunity in either a positive or negative manner, depending upon their lineage and state of maturation. There are several subpopulations of DC including myeloid DC (mDC), plasmacytoid DC (pDC), and Langerhans cells (LC) that play different roles in the regulation of the immune responses. In addition to their ability to stimulate immunity, these different DC populations, under certain conditions, are involved in T cell immunosuppression and/or induction of central and peripheral tolerance. We and others have demonstrated that systemic administration of bone marrow (BM)-derived, myeloid DC, genetically modified to express either IL-4 or Fas ligand (L), is able to reverse established murine autoimmune arthritis for extended periods of time following a single treatment. Exosomes are small membrane vesicles, 40 to 100 nm in size, released by various cell types through the endocytic pathway. Exosomes from APC carry MHC I and II and T cells costimulatory molecules on their surface, suggesting that they could play important roles in immune regulation. In murine models, DC-derived exosomes have been shown be immunostimulatory or suppressive, depending on the type and stage of maturation of the DC. We demonstrated that both DC and exosomes derived from immature DC, pre-treated with IL-10, produce anti-inflammatory exosomes that suppress the onset of murine CIA and reduce the severity of established arthritis. In fact, exosomes were as effective as the parental DC in suppressing CIA onset. Moreover, DC transduced with recombinant adenovirus encoding FasL (Ad.FasL) produce exosomes able to suppress inflammation in a model of delayed type hypersensitivity (DTH) and partially reverse established CIA in mice. The ability of the Ad.FasL transduced DC and DC/FasL- derived exosomes to suppress the DTH response was dependent not only upon FasL in the DC or DC-derived exosomes, but also on the presence of Fas in the host mice. Moreover, the effect was MHC class II dependent, but MHC Class I independent. We currently are examining the mechanisms through which DC and DC-derived exosomes are able to suppress the immune response in murine models of diabetes, arthritis and EAE. In addition, we are examining the ability of tumor-derived exosomes to modulate the anti-tumor response. 

Gene Therapy for Cancer: Interleukin-12 is a heterodimeric cytokine produced by monocytes important for stimulating NK and T cells in vivo as well as stimulating differentiation of TH1 T-cells. We have developed retroviral vectors and more recently adenoviral vectors expressing both subunits of mouse and human interleukin-12. These vectors have been used in mouse tumor models to demonstrate a therapeutic, anti-tumor effect of local interleukin-12 expression. These results have led to submission, approval, and initiation of a clinical protocol for cancer using gene transfer of interleukin-12. The current focus of the lab is to determine if the antitumor effects of IL-12 can be stimulated through the use of other immunostimulatory protein including cytokines (i.e. GM-CSF, IL-10, IL-18), cell surface molecules (i.e. B7.1, CD40L, CD27L) and apoptosis/necrosis-inducing proteins (i.e. p53, HSV tk). We also are examining the ability of IL-12 family members, IL-23 and IL-27, to confer anti-tumor effect either alone or in combination. 

Regulation by SIRT1: hSir2, also known as SIRT1, is the human homolog of the yeast Silence Information Regulator (Sir2) gene. It is a NAD+ dependent deacetylase (class III HDAC) that belong to the sirtuin family. SIRT proteins are known to play a role in a wide range of cellular processes such as transcriptional regulation, cell-division cycle, microtubule function, muscle differentiation, early embryogenesis, cellular stress response and molecular mechanisms of aging. However the intricate mechanism by which SIRT1 mediates its role in cell survival and longevity remains largely elusive. SIRT1 has been shown deacetylates histone proteins and certain transcription factors such as p53, CTIP2, FOXO and NF-ĸB. To identify potential SIRT1 interacting factors, we performed a yeast two-hybrid screen. The screen identified Transducin like Enhancer of split 1 (TLE1) as a possible SIRT1-interacting factor which was then confirmed by co-immunoprecipitation. TLE1 is a non-DNA binding co-repressor for several transcriptional factors including NF-ĸB. These results suggest that the interaction between SIRT1 and TLE1 is important for mediating repression of NF-ĸB activity. We also identified EIF2-alpha as a factor associating with SIRT1 in the two hybrid screen, which was confirmed by co-IP. Thus we have examined the ability of SIRT1 to regulate translation. Our data from SIRT1 -/- MEFs and Hela cells that are reduced in siRNA, demonstrated difference in the level of mTOR in wt versus SIRT1 knock-down cells. Since SIRT1 is already known to play a role in cellular stress response; we looked at the levels of several proteins that are involved in the translation regulation, in response to a variety of stress signals such as low serum, low nutrient and ER stress. Our results suggest that a number of proteins in the eIF2-alpha and mTOR stress signaling pathway are regulated by SIRT1. Since translation regulation is one of the first steps of cellular response to stress, and we have observed that SIRT1 regulates a number of different translation regulatory proteins involved in cell survival and size, we are currently examining further the potential role of SIRT1 in translation regulation. 

Role of NF-κB and Stem Cells in the Molecular and Cellular Basis of Age-Related Degeneration: The loss of tissue homeostasis is generally accepted to arise as a consequence of the time-dependent accumulation of cellular damage, including DNA damage. Furthermore, chronic inflammation and, in particular, oxidative stress are implicated in many age-related degenerative diseases and likely cause much of this cellular damage. What is not known is the precise mechanism through which the accumulation of cellular damage promotes tissue degeneration. A key mediator of the cellular stress response and inflammation is the transcription factor NF-κB. The activity of NF-κB is up-regulated in response to different types of cellular stress and in tissues of aged organisms, making it an excellent candidate mediator of age-related degenerative changes. Genetic and pharmacologic reduction in the level of the NF-κB subunit p65(RelA) progeria mouse models of accelerated aging delayed the onset of age-related pathology including osteoporosis, sarcopenia, intervertebral disc degeneration, loss of islets and neurodegeneration. We currently are testing the hypothesis that accumulation of damage caused by oxidative stress induces NF-κB transcriptional activity, in certain cell types including stem cells, which then regulates proliferation, cellular senescence, apoptosis and differentiation and promotes degenerative diseases. 

Facilitating bone healing using Lim Mineralization Protein: Although the delivery of BMPs as recombinant proteins can induce local bone formation and healing of bone defects, it has been demonstrated that local gene transfer of BMP-2 at the site of critical size femoral and cranial defects in the rabbit and rat resulted in more rapid and efficient bone healing. More recently, we and others have shown that gene transfer of the LIM Mineralization Protein (LMP), a novel intracellular positive regulator of the osteoblast differentiation program, can induce efficient bone formation. In humans, three different LMP splice variants have been identified, termed LMP-1, LMP-2, and LMP-3. Gene transfer of human LMP-1 and LMP-3 induces expression of genes involved in bone formation including certain bone morphogenetic proteins (BMPs), promotes bone nodule formation in vitro, ectopic bone formation in vivo, healing of critical size defects and can facilitate posterior thoracic and lumbar spine fusion. We have demonstrated that gene transfer of the hLMP-3 isoform is able to induce bone mineralization, expression of the bone-specific genes in cell culture and ectopic bone formation in mouse muscle and healing of rat segmental and mandibular bone defects in vivo. We now have mapped the osteoinductive effects of LMP-3 to a 20 amino acid or smaller region in LMP-3, which we have termed Osteoinductive Domain-1 (OD-1), able to induce mineralization and bone specific gene expression in cell culture and confer induction of ectopic bone formation in vivo. Currently we are examining the mechanism(s) through which this osteoinductive domain functions to mediated osteogenesis.

As of July 1, 2012, Dr. Robbins accepted a position at The Scripps Institute in Juniper, FL. We wish Dr. Robbins the best of luck in his future endeavors.