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Paul D. Robbins, Ph.D.
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Professor
W1246 BSTWR
200 Lothrop Street
Pittsburgh, Pennsylvania 15261
Phone: (412) 648-9268
Fax: (412) 383-8837
E-mail: probb@pitt.edu
Lab Phone: (412) 648-9268
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Biography
Dr. Paul D. Robbins is a Professor of Microbiology and Molecular Genetics and Orthopaedic Surgery at the University of Pittsburgh School of Medicine. He is also Director of the Vector Core Facility. He received his B.A. from Haverford College and his Ph.D. from the University of California at Berkeley and worked as a post-doctoral fellow at the Whitehead Institute for Biomedical Research at the Massachusetts Institute of Technology. He is an Associate Editor for Cancer Research and Gene Therapy as well as on the Editorial Boards for Cancer Gene Therapy, The Journal of Gene Medicine, Arthritis Research, and Genes & Immunity. Dr. Robbins has co-authored over 230 peer-reviewed manuscripts and 130 book chapters and reviews and has edited two books on gene therapy. He was a member of the PathB Study Section, and was Chair of the Italian Telethon Scientific Review Committee, a former member of the Board of Directors of the American Society of Gene Therapy as well as former Chair of the American Society of Gene Therapy Committee on Musculoskeletal Diseases. He also is a member of the Scientific Advisory Boards for Tissuegene, Orthogen and Viromed.
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Research
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.
Gene Therapy to Facilitate Islet Transplantation: Insulin-dependent diabetes mellitus (IDDM) is characterized by destruction of the insulin-producing pancreatic ß-cell. Analysis in the nonobese diabetic (NOD) mouse model of autoimmune diabetes suggests that the destruction of islets during the course of disease may be caused by one or more of three distinct death effect systems, Fas/FasL, perforin, and TNF/TNF-receptor. However, recent data suggests that the Fas-FasL interaction is the predominant pathway for ß-cells destruction during progression of disease. Indeed, Fas is upregulated by IL-1ß during the early stages of IDDM, resulting in apoptosis of the ß-cells specifically through a NO dependent mechanism. Currently there is no effective form of treatment for IDDM except for multiple daily injections of insulin or by transplantation of allogeneic or xenogeneic islets. Multiple approaches to facilitate islet transplantation have been attempted including treatment with immunosuppressive agents such as FK506 and cyclosporin as well as encapsulation of the islets in different immunoisolation matrices. In addition, gene transfer of immunosuppressive agents to either islets or cells coadmixed with allogeneic islets has partially prolonged engraftment in murine models. We have shown that both human and murine islets can be efficiently infected with lentiviral, AAV and adenoviral vectors. Furthermore, the local expression IL-1 inhibitors in islets such as IL-1Ra is able to block IL-1 mediated islet dysfunction and Fas-mediated apoptosis. We are now examining the ability of different gene products (CTLA4-Ig, PD-L1, sCD40-Ig, IkB, etc) expression in transplanted syngeneic islets to block apoptosis in the NOD mouse and are attempting to block allogeneic islet rejection using transfer to islets of genes encoding immunosuppressive factors.
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.
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Selected Publications
- Bianco NR, Kim SH, Ruffner MA, Robbins PD. (2009) "Therapeutic effect of exosomes from indoleamine 2,3-dioxygenase-positive dendritic cells in collagen-induced arthritis and delayed-type hypersensitivity disease models" Arthritis Rheum. 60:380-9. | Abstract
- Rehman KK, Trucco M, Wang Z, Xiao X, Robbins PD. (2008) "AAV8-mediated gene transfer of interleukin-4 to endogenous beta-cells prevents the onset of diabetes in NOD mice" Mol Ther. 16:1409-16. | Abstract
- Sun F, Mi Z, Condliffe SB, Bertrand CA, Gong X, Lu X, Zhang R, Latoche JD, Pilewski JM, Robbins PD, Frizzell RA. 2008. "Chaperone displacement from mutant cystic fibrosis transmembrane conductance regulator restores its function in human airway epithelia" FASEB J. 22:3255-63. | Abstract
- Davé SH, Tilstra JS, Matsuoka K, Li F, Karrasch T, Uno JK, Sepulveda AR, Jobin C, Baldwin AS, Robbins PD, Plevy SE. 2007. "Amelioration of chronic murine colitis by peptide-mediated transduction of the IkappaB kinase inhibitor NEMO binding domain peptide" J Immunol. 179:7852-9. | Abstract
- Kim SH, Bianco NR, Shufesky WJ, Morelli AE, Robbins PD. (2007) "Effective treatment of inflammatory disease models with exosomes derived from dendritic cells genetically modified to express IL-4" J Immunol. 179:2242-9. | Abstract
- Rehman KK, Bertera S, Trucco M, Gambotto A, Robbins PD. 2007. "Immunomodulation by adenoviral-mediated SCD40-Ig gene therapy for mouse allogeneic islet transplantation" Transplantation 84:301-7. | Abstract
- Ghosh HS, Spencer JV, Ng B, McBurney MW, Robbins PD. 2007. "Sirt1 interacts with transducin-like enhancer of split-1 to inhibit nuclear factor kappaB-mediated transcription" Biochem J. 408:105-11. | Abstract
- Niedernhofer LJ, Robbins PD. 2008. "Signaling mechanisms involved in the response to genotoxic stress and regulating lifespan" Int J Biochem Cell Biol. 40:176-80. | Abstract
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