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323. Liver-Directed, Insulin-Based Gene Therapy Protects NOD Mice from Diabetes
PRESIDENTIAL SYMPOSIUM we mixed equal numbers of U87 cells expressing the ten different Gluctag and implanted the heterogeneous cell pool subcutaneously in nude mice. We monitored tumor growth by Fluc bioluminescence imaging and total Gluc blood assay. Importantly, we were able to efficiently monitor the growth of the individual subpopulation of the U87 cells expressing the different Gluctag reporters in the same tumor over time. We confirmed the expression of all tags in the same tumor by immunostaining. To determine the applicability of the Gluctag multiplex system in deep tissues, the mixture of the ten different U87FM-Gluctag-CFP cells were implanted orthotopically in the brain of nude mice. We monitored tumor growth by Fluc bioluminescence imaging and total Gluc blood assay. Again, we were able to monitor the intracranial growth of the individual subpopulations of U87 cells expressing the Gluctag reporters within the same brain tumor in realtime, by blood sampling and the application of our Gluctag multiplex reporter system. We now aim to use our Gluc-tag multiplex system to analyse transcription factors (TF) and its resulting gene expression profile. We constructed a library of lentiviral vectors consisting of the Gluc cDNA fused to one of six different epitope tags (Flag, His, Ha, AcV5, V5 and Glu) at its C-terminus, under TF specific promoters resulting in an array of six TF-Gluc-tag reporters. We employed U87 cells for validation of the TF-Gluc-tag reporter multiplex system. These cells were transduced with each of the lentiviral vectors to produce six different cell lines, each stably expressing a different TF-Gluc-tag reporter. Using TF expression vectors will validate the TF activity reporters and aim to use these reporters to analyse biologically relevant treatments of glioblastoma cells and crosslink the TF activity profiles with the glioblastoma gene expression arrays to determine relevant gene expression pathways involved.
Presidential Symposium 322. Kinetics and Epigenetics of Retroviral Silencing in Mouse Embryonic Stem Cells Defined by Deletion of a D4Z4 Barrier Element
Sylvie Rival-Gervier,1 Mandy Lo,1 Shahryar Khattak,1 Peter Pasceri,1 Matthew Lorincz,2 James Ellis.1 1 Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, ON, Canada; 2Medical Genetics, University of British Columbia, Vancouver, BC, Canada. Retroviral vectors are silenced by epigenetic mechanisms in embryonic stem (ES) cells that cannot be fully prevented and whose kinetics are poorly understood. The HSC1 self-inactivating retroviral vector is silenced in mouse ES cells rapidly after integration or over prolonged culture, even when favourable integration sites for transgene expression are selected by cell sorting or drug selection. To escape this silencing, we tested the D4Z4 insulator. We show that a 3’D4Z4 fragment directs retroviral expression in ES cells with persistent but variable expression for up to 5 months. Combining an internal 3’D4Z4 with HS4 insulators in the LTRs shows that these elements cooperate, and defines the first retroviral vector that fully escapes long-term silencing. Epigenetic analyses demonstrate that 3’D4Z4 and HS4 insulators together maintain hypomethylated DNA on retroviral transgenes and reduce H3K9me3 repressive marks to the same level as the expressed endogenous Nanog locus. In order to decipher the role of 3’D4Z4 activity at specific integration sites, we used FLP recombinase to induce deletion of 3’D4Z4 from ES cell clones that contain a single provirus. We observe that after 3’D4Z4 deletion retroviral silencing is established at many but not all integration sites. This finding shows that 3’D4Z4 does not target retrovirus integration into favourable epigenomic domains. Instead, epigenetic analyses demonstrate that 3’D4Z4 is a barrier element that blocks the spread of heterochromatin marks including DNA methylation and repressive histone modifications such as H3K9 methylation. In addition, our deletion system reveals three distinct S124
kinetic classes of silencing (rapid, gradual or not silenced), and our epigenetic analyses suggest that multiple pathways participate in silencing at different integration sites. We conclude that vectors with both the 3’D4Z4 barrier and HS4 insulator elements block rapid and gradual kinetic classes of silencing, and may have unprecedented utility for gene transfer applications that require long-term gene expression in pluripotent stem cells. This vector is well suited for manipulation of iPS cells for genetic rescue in disease modeling studies and has potential for suicide vector applications to increase the safety of iPS or ES cell therapies.
323. Liver-Directed, Insulin-Based Gene Therapy Protects NOD Mice from Diabetes
Mahzad Akbarpour,1,3 Kevin S. Goudy,1 Andrea Annoni,1 Francesca Sanvito,2 Luigi Naldini,1,3 Maria Grazia Roncarolo.1,3 1 Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Telethon Institute for Gene Therapy (OSR-TIGET), Milan, Italy; 2Division of Molecular Oncology, San Raffaele Scientific Institute, Milan, Italy; 3Vita-Salute San Raffaele University, Milan, Italy. Immunotherapy is an ideal approach to induce antigen (Ag)-specific tolerance in T cell-mediated autoimmune diseases, such as type 1 diabetes (T1D). In order to achieve tolerance using this approach, the Ag must be presented in a context that depletes pathogenic effector T cells (Teff) and/or promotes the induction of regulatory T cells (Tregs). The insulin B peptide 9-23 (InsB) is an auto-Ag in human and non-obese diabetic mice (NOD) model of human T1D that is targeted by T cells. The expression of auto-Ags in the liver is known to drive Ag-specific tolerogenic responses. To determine if InsB expression in the liver can prevent T1D in NOD mice we generated a lentiviral vector (LV) containing a hepatocyte specific promoter (ET) and miR-142 target sequences (142T) to selectively target InsB expression to hepatocytes. Pre-diabetic 11wk-old female NOD mice were treated with ET.InsB.142T (LV.InsB) and LV encoding a control Ag ovalalbumin (OVA) ET.OVA.142T (LV. OVA) and followed for diabetes development. LV.InsB treated NOD were protected (90%) from diabetes development at 43 weeks of age compared to control mice left untreated or treated with LV.OVA. Treatment of NOD mice with LV.InsB increases FoxP3+Tregs in the liver and pancreatic draining lymph node, maintains transgene expression in hepatocytes and reduces diabetes incidence despite inducing InsB-specific, IFN-producing CD8+ T cells. Histological analysis showed that the LV.InsB treatment reduced the severity of islet infiltration and preserved insulin production compared to LV.OVA treated NOD, immunohistochemistry analysis of the islet infiltrating cells in the protected revealed the presence of FoxP3+ T cells. Gene expression of cells in the pancreatic draining lymph nodes 7 weeks after LV.InsB treatment revealed that in addition to Foxp3, the level of tolerogenic molecules il10, pdl-1 and tgfb is increased compared to age matched LV.OVA treated. Adoptive transfer of splenocytes from protected LV.InsB treated mice prevented diabetes transfer to recipient NOD-Scid mice; while LV.OVA treated mice and Treg depleted splenocytes form protected InsB-treated mice did not. These data suggest that T1D prevention in this model occurs in an Ag-specific, Treg mediated manner since splenocytes from LV.OVA treated and Treg depleted LV.InsB treated mice failed to establish tolerance in recipient NOD-Scid mice. Furthermore, to reduce the potential risk of insertional mutagenesis experiments performed using integrase-defective lentiviral vector (IDLV) with the same expression cassettes. Hepatocyte-directed IDLV.InsB, also effectively prevented T1D, demonstrating that the restricted expression of an auto-Ag in the liver, even for a short duration of expression, can induce Ag-specific Tregs that can control autoimmunity residing in another anatomic location. These findings present a promising therapeutic advance in designing gene delivery systems that target the liver to induce tolerance in T1D and other T-cell mediated diseases. Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
Presidential Symposium 322. Kinetics and Epigenetics of Retroviral Silencing in Mouse Embryonic Stem Cells Defined by Deletion of a D4Z4 Barrier Element
Sylvie Rival-Gervier,1 Mandy Lo,1 Shahryar Khattak,1 Peter Pasceri,1 Matthew Lorincz,2 James Ellis.1 1 Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, ON, Canada; 2Medical Genetics, University of British Columbia, Vancouver, BC, Canada. Retroviral vectors are silenced by epigenetic mechanisms in embryonic stem (ES) cells that cannot be fully prevented and whose kinetics are poorly understood. The HSC1 self-inactivating retroviral vector is silenced in mouse ES cells rapidly after integration or over prolonged culture, even when favourable integration sites for transgene expression are selected by cell sorting or drug selection. To escape this silencing, we tested the D4Z4 insulator. We show that a 3’D4Z4 fragment directs retroviral expression in ES cells with persistent but variable expression for up to 5 months. Combining an internal 3’D4Z4 with HS4 insulators in the LTRs shows that these elements cooperate, and defines the first retroviral vector that fully escapes long-term silencing. Epigenetic analyses demonstrate that 3’D4Z4 and HS4 insulators together maintain hypomethylated DNA on retroviral transgenes and reduce H3K9me3 repressive marks to the same level as the expressed endogenous Nanog locus. In order to decipher the role of 3’D4Z4 activity at specific integration sites, we used FLP recombinase to induce deletion of 3’D4Z4 from ES cell clones that contain a single provirus. We observe that after 3’D4Z4 deletion retroviral silencing is established at many but not all integration sites. This finding shows that 3’D4Z4 does not target retrovirus integration into favourable epigenomic domains. Instead, epigenetic analyses demonstrate that 3’D4Z4 is a barrier element that blocks the spread of heterochromatin marks including DNA methylation and repressive histone modifications such as H3K9 methylation. In addition, our deletion system reveals three distinct S124
kinetic classes of silencing (rapid, gradual or not silenced), and our epigenetic analyses suggest that multiple pathways participate in silencing at different integration sites. We conclude that vectors with both the 3’D4Z4 barrier and HS4 insulator elements block rapid and gradual kinetic classes of silencing, and may have unprecedented utility for gene transfer applications that require long-term gene expression in pluripotent stem cells. This vector is well suited for manipulation of iPS cells for genetic rescue in disease modeling studies and has potential for suicide vector applications to increase the safety of iPS or ES cell therapies.
323. Liver-Directed, Insulin-Based Gene Therapy Protects NOD Mice from Diabetes
Mahzad Akbarpour,1,3 Kevin S. Goudy,1 Andrea Annoni,1 Francesca Sanvito,2 Luigi Naldini,1,3 Maria Grazia Roncarolo.1,3 1 Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Telethon Institute for Gene Therapy (OSR-TIGET), Milan, Italy; 2Division of Molecular Oncology, San Raffaele Scientific Institute, Milan, Italy; 3Vita-Salute San Raffaele University, Milan, Italy. Immunotherapy is an ideal approach to induce antigen (Ag)-specific tolerance in T cell-mediated autoimmune diseases, such as type 1 diabetes (T1D). In order to achieve tolerance using this approach, the Ag must be presented in a context that depletes pathogenic effector T cells (Teff) and/or promotes the induction of regulatory T cells (Tregs). The insulin B peptide 9-23 (InsB) is an auto-Ag in human and non-obese diabetic mice (NOD) model of human T1D that is targeted by T cells. The expression of auto-Ags in the liver is known to drive Ag-specific tolerogenic responses. To determine if InsB expression in the liver can prevent T1D in NOD mice we generated a lentiviral vector (LV) containing a hepatocyte specific promoter (ET) and miR-142 target sequences (142T) to selectively target InsB expression to hepatocytes. Pre-diabetic 11wk-old female NOD mice were treated with ET.InsB.142T (LV.InsB) and LV encoding a control Ag ovalalbumin (OVA) ET.OVA.142T (LV. OVA) and followed for diabetes development. LV.InsB treated NOD were protected (90%) from diabetes development at 43 weeks of age compared to control mice left untreated or treated with LV.OVA. Treatment of NOD mice with LV.InsB increases FoxP3+Tregs in the liver and pancreatic draining lymph node, maintains transgene expression in hepatocytes and reduces diabetes incidence despite inducing InsB-specific, IFN-producing CD8+ T cells. Histological analysis showed that the LV.InsB treatment reduced the severity of islet infiltration and preserved insulin production compared to LV.OVA treated NOD, immunohistochemistry analysis of the islet infiltrating cells in the protected revealed the presence of FoxP3+ T cells. Gene expression of cells in the pancreatic draining lymph nodes 7 weeks after LV.InsB treatment revealed that in addition to Foxp3, the level of tolerogenic molecules il10, pdl-1 and tgfb is increased compared to age matched LV.OVA treated. Adoptive transfer of splenocytes from protected LV.InsB treated mice prevented diabetes transfer to recipient NOD-Scid mice; while LV.OVA treated mice and Treg depleted splenocytes form protected InsB-treated mice did not. These data suggest that T1D prevention in this model occurs in an Ag-specific, Treg mediated manner since splenocytes from LV.OVA treated and Treg depleted LV.InsB treated mice failed to establish tolerance in recipient NOD-Scid mice. Furthermore, to reduce the potential risk of insertional mutagenesis experiments performed using integrase-defective lentiviral vector (IDLV) with the same expression cassettes. Hepatocyte-directed IDLV.InsB, also effectively prevented T1D, demonstrating that the restricted expression of an auto-Ag in the liver, even for a short duration of expression, can induce Ag-specific Tregs that can control autoimmunity residing in another anatomic location. These findings present a promising therapeutic advance in designing gene delivery systems that target the liver to induce tolerance in T1D and other T-cell mediated diseases. Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy